Acta Physiol Scand 1979, 106: 199-214
Principal mechanisms controlling penile retraction and protrusion in rabbits NILS 0. SJOSTRANDand ERIK KLINGE Department of Physiology, Karolinska Institutet, Stockholm, Sweden, and the Department of Pharmacology, University of Helsinki, Finland
SJOSTRAND, N. 0. & KLINGE, E.: Principal mechanisms controlling penile retraction and protrusion in rabbits. Acta Physiol Scand 1979, 106: 199-214. Received 30 Nov. 1978. ISSN 00016772. Dept. of Physiology, Karolinska Inst. and Dept. of Pharmacology, University of Helsinki, Finland. The effects on penile volume of nerve stimulations and drugs injected into the systemic circulation were studied plethysmographically. Dilator responses at selective perfusion of the penile artery were studied by measuring the perfusion pressure. The main results and conclusions are: The penis has an adrenergic vasoconstrictor supply coming from the sacrococcygeal parts of the sympathetic chains. A very low (0.2 Hz) vasomotor tone keeps the penis relaxed. If this tone is interrupted the penis will protrude but autoregulation will soon take over and eventually produce hyperinvolution of the penis. Two vasodilator paths, both with pelvic ganglionic relays, were found. I ) The pelvic parasympathetic nerves, probably having mainly non-cholinergic postganglionic neurons and operating quite effectively at low frequencies. 2) The sympathetic hypogastric nerves, presumably having at least partly cholinergic postganglionic neurons which, apart from muscarinic dilation of minute inflow resistance vessels to the erectile tissue, may also work by suppression of excitatory adrenergic neurotransmission. The pelvic and hypogastric vasodilator outflows work synergistically. The vasoconstrictor nerves are very strong and efficient antagonists of the vasodilator nerves. Key words: Penis, erection, autonomic innervation, vasodilation, smooth muscle
A primary event in mammalian penile erection is dilation of the arterial branches forming the “inflow resistance” vessels to the cavernous bodies. This leads to a situation where the “outflow resistance” exceeds the inflow resistance and consequently the erectile tissue will be engorged. Conversely, vasoconstriction forms the basis of the flaccid state of the penis. The nature of penile vasodilation has been a matter of dispute for a long time (vide Klinge & Sjostrand 1974). Two main issues have been debated. 1) Are the pelvic parasympathetic nerves, i.e. the nervi erigentes of Eckhard (1863), the only path for penile vasodilator fibres? This seems to be the predominant view apparently based on the works of Langley & Anderson (1895) and Semans & Langworthy (1938) where other routes are refuted. However, there are continental reports indicating that erector fibres also could travel along the sympathetic system, especially the hypogastric nerves (Eckhard 1876, FranGois-Franck 1895, Bacq 1935,
Bessou & Laporte 1961, Thielen, Renders & Rectern 1969). 2) Are the dilator fibres cholinergic, as generally assumed, or not? In fact, this question is much older than the concept of cholinergic nerves and goes back to a dispute on whether atropine “paralyzes” (Nikolsky 1879) the nervi erigentes or not (Anrep & Cybulski 1884). In this century a cholinergic transmission of erection has been proposed by e.g. Henderson & Roepke (1933), Bacq (1935). Oppenheimer (19381, Bessou & Laporte (1961) and Hukovic & Bubic (1%7), while it has been doubted by e.g. Luduena & Grigds (1966) and Dorr & Brody (1967). We have criticized the pharmacological evidence presented by earlier investigators, the main objections being the combined use of preganglionic stimulation and very high frequencies which make it difficult to evaluate the point of attack and the effect of drugs such as atropine and physostigmine (Klinge & Sjostrand 1974). Concerning the putative sympathetic erector fibres there is also an objection Actti
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against the use of a-adrenergic blocking ergot compounds (Bacq 1935, Bessou & Laporte 1961) in order to make the vasodilation clear, because the observed effect could be d u e t o adrenergic p-receptors. Experiments on various isolated smooth muscle effectors of penile erection have led us to the conclusion that neurogenic relaxation of these smooth muscles is hardly cholinergic although cholinergic nerves may contribute t o erection by prejunctional inhibition of excitatory adrenergic neurotransmission to these effectors (Klinge & Sjostrand 1974, 1977ri and b ) . In this report we present results from in vivo studies performed with the aim to obtain information on the basic myogenic and neurogenic control mechanisms of penile volume. The rabbit, having a penis rather similar to that of man (Fujimoto & Takeshige 197% was chosen as experimental animal. A preliminary account of the results has been presented (Sjostrand & Klinge 1978).
MATERIAL A N D M E T H ODS Animuls
This and the subsequent report (Sjostrand & Klinge 1979) are based on expts. on 58 rabbits of different breeds; 32 were albinos of the New Zealand race or native strains. B.wt. was in the range of 2.0-5.5 kg. Operrrtiw procedures
I . Generul prepurution und care. The rabbits were starved for 12-18 h and anesthetized with urethan (16-20 mmol/kg i.v.). They were tracheostomized and a T-tube was inserted. The abdomen was opened with a midline incision. The inferior and superior mesenteric arteries and the corresponding branches of the portal vein were tied and the gut was removed between the duodenum and the oral end of the rectum. The ureters were cannulated and the bladder was emptied. The vessels of the bladder were tied and the bladder was resected above the trigonum area. A cotton-wool pellet soaked in vaseline was introduced into the urethra through the opened bladder and placed at the level of the pubic bone in order to prevent secretion from the male sex accessories into the penis. The vasa deferentia were ligated in the ampullary region and the seminal vesicle was slit in its tips and the secretion was removed. A catheter for i.v. infusions was introduced into either a femoral or an external jugular vein. In some expts. a catheter for i.a. injections was introduced into a femoral or a subclavian artery and pushed to the aortic origin of the vessel. Arterial blood pressure was recorded from one of the carotid arteries (usually the left) or a femoral artery. I n expts. in which the sympathetic chains were cut and stimulated the external iliac vessels were usually tied in order to prevent drop of blood pressure and pooling of blood in the hind legs. In some expts. artificial Avfrr Ph.wio/ Sc~rnd/IN
ventilation was given under decarnethonium ( 1.1-2.4 pmol/kg i.v.) blockade. After evisceration the animals received 10 ml 6 9i dextran in saline (Macrodex@,Pharmacia) per kg b.wt. During the course of the expt. the rabbits received 2-6 rnl of each of the following solutions per h: glucose-fructohr (both 0.56 M), NaHCO, 1.6x lo-' M. 6 % dextran and Ringer or saline. 2. Plethysmogrriphy ofthe penis. The attachment of the penile skin to the rectum was severed. The opening in the penile skin as well as that in the rectal skin were firmly closed by sutures. The preputium was elevated and its inner layer was separated from the penis by a circular incision through the attachment beneath the glans penib. Via this incision the entire penis was stripped by blunt dissection. The skin tube covering the penis was left in continuity with the pubic and the perineal skin. The skinned penis was lubricated with paraffin oil and a greased glass plethysmograph provided with 3 hooks was introduced into the skin tube and placed around the mentulum. The plethysmograph was resting on the dome formed by the perineal striated muscles encircling the crura and the bulb of the penis. Ligatures from the penile skin to the hooks kept the plethysmograph in place and complete sealing was achieved by tying threads around the base of the penile skin and plethysmograph (Fig. I ) . The recording system was tilled with 7-8 ml of air and care was taken to keep the room temperature constant. 3. Selective perfusion of the penile urtery. The penis. the pubic, perineal and sacrococcygeal regions as well as the hips were flayed. Via blunt dissection in the genitofemoral groove, cutting off covering muscles and removing the tuber ossis ischii with its muscular insertions the internal pudic artery was identified in "Alcock's canal". In the 5 New Zealand rabbits used in this type of expt. there was quite a difference in size of the penile arteries on the two sides. The smaller artery was ligated while the larger was cannulated at its origin, i.e. where the terminal pudic artery divides into the penile and rectal branches. All visible penile veins were ligated centrally and cut peripherally and the tip of the glans penis was cut off to permit outflow of perfusion fluid. The penile artery was perfused with Tyrode solution (Klinge & Sjostrand 1974) which contained 2 % FicolP (Pharmacia), M.w. about 77000. and 1.5% FicolP, M.w. about 47000, as colloid and was aerated with 6S%, CO, in 0, and kept at about 34°C. Perfusion rate was 1.75-3.0 ml/min and histamine (0.S5.0x lo-' M) was added to induce sufficient vascular tone. i.e. an effective initial perfusion pressure close to 100 mmHg. The pressure was measured via a T-tube inserted into the arterial cannula. An inlet for injections was placed between the reservoir and the pump close to the latter. 4 . Dissection and stimulation of nerves. Good descriptions of the pelvic, hypogastric and pudic nerves in the rabbit and of the approach to them are given by Langley & Anderson (1895, 18%). These guidelines were essentially followed. The lumbosacral sympathetic chains were identified and prepared at the level of LBS1. The left splanchnic nerve was isolated rather close to the adrenal. In three expts. in which adrenomedullary stimulation was performed the right adrenal was separated from the circulation by tight ligatures. The vagus nerves were isolated a1
SKIN
PLETHYSMOGRAPH PENIS
LIGATURES
Fig. 1 . Schematic drawing of the glass plethysmograph in
situ. Frontal view. The plethysmograph is connected via a low pressure transducer to a Grass polygraph. The degree of protrusion of the rabbit penis, which is retracted in the above illustration, is also directly visible. For further explanations see "Material and Methods".
about the level of the thyroid cartilago. The cut nerves were placed on hook-shaped platinum electrodes ( 2 mm apart) shielded underneath with plexiglass and provided on the upper side with a movable plexiglass rod for fixing the nerve, All electrodes were provided with holders designed specially for each nerve. The holders were fixed to the operation table in order to keep the electrodes in position during the expt. Paraffin oil in ample amount was used for isolation. The nerves were stimulated with square wave pulses (4-6 ms) and "supramaximal" voltage (15-25 V). Unless otherwise stated, all stimulations were bilateral. The usual stimulation period was 20 s . This will be referred to as standard stimulation. In some frequency-response expts. we stimulated until the response became stabilized at a fixed level. This will be referred to as stimulation with unlimited train. 5. Section of lutnbosacrul sympathetic chains prior to expts. Six rabbits were pretreated with promethazine (16 pmol/kg i.m.) and anesthetized with sodium pentobarbital (120-200 pmol/kg i.v.). The abdomen was opened along the linea alba and the paravertebral chains were dissected free around the ganglia at L,. The cranial and caudal connections of these ganglia and those at L,, and S, were removed. whereafter the abdomen was closed. The animals were used for expts. 2. 3, 4, 6, 14 or 21 d after operation. Special considerations in the unalysiu of the effects of antirnuscarinic drugs on erectile responses
The use of antimuscarinic drugs as atropine or scopolamine in in vivo expts. on rabbits is encumbered with many drawbacks. Many rabbits have an inherited atropinesterase in plasma, liver and several other organs, which rapidly inactivates both atropine and scopolamine (for ref. see e.g. Kalow 1%2, Cauthen et al. 1976). There further is the problem of reaching a sufficient concentration of drugs in a normally poorly perfused organ as the penis. Therefore all effects of atropine and scopolamine on the response of the penis to pelvic or hypogastric nerve stimulation were evaluated against the effect of the drugs on the response of the heart to stimulation on the right vagus nerve (20 s , 15-25 Hz). This response is one of the most atropine
sensitive cholinergic nerve effects (e.g. Ursillo 1961). Consequently only in expts. where this effect could be blocked one might obtain significant pharmacological data on the possible muscarinic nature of the erectile response. We could divide our rabbits into 3 groups: a small group (only 3 rabbits) in which the vagal bradycardia could be blocked for several h by moderate doses (4-20 pmol/kg) of the drugs, a main group in which just a short-lived (3-15 min) suppression of the bradycardia was obtained with even huge doses (70-170 pmol/kg) of the alkaloids, and finally one group where even grotesque doses (more than 216 pmol/kg) were without any effect. These groups were defined as atropine sensitive. semiresistant and resistant, respectively. The difficulties in evaluating the effects of the antimuscarinic drugs prompted us to do the perfusion expts. Atropine and scopolamine also have ganglionic blocking properties (vide Klinge & Sjostrand 1974) which could impair the analysis. This possibility was checked by recording contractions of the seminal vesicle to injected noradrenaline and to hypogastric nerve stimulation which is preganglionic to that organ (vide Sjostrand 1965). We obtained no clear-cut evidence for a ganglionic action of the alkaloids when doses of 4-40 pmol/kg were used. However. with the higher doses. which we had to use in the perfusion expts. and also in the expts. on atropine sensitive rabbits, an interference of a ganglionic or postganglionic nicotinic point of attack cannut be excluded. Equipment Recordings were made on a Grass polygraph. Statham transducers were used for pressure recordings and a Grass volumetric low-pressure transducer (PT5) was used in plethysmography. A Sigma motor pump was used in the perfusion expts. Grass S4, S44, S5 or SD 9 stimulators delivered the shocks. Slow infusions were made with a Braun infusion pump. Temperatures were measured with Gold Brand precision thermometers and a Harvard respirator was used for artificial ventilation. Some critical comments on the tnethodologv
During evisceration the rectal temperature fell to 35-33°C and stayed at this level throughout the expt. despite warming. Together with the damping of the recording system this means that our quantitative data are sluggish compared to those of "real life". The airfilled system in plethysmography had of course the errors of this method. Nervous injuries due to preparation should not be overlooked. Especially the pelvic nerves were difficult to prepare satisfactorily in contrast to the easy hypogastric nerves. Drugs The following pure salts, bases and peptides were used: I-noradrenaline~-bitartrate, I-adrenaline-d-bitartrate, terbutaline sulphate, phenoxybenzamine hydrochloride, phentolamine methane sulphonate, propranolol hydrochloride, guanethidine sulphate, acetylcholine chloride, neostigmine bromide, atropine sulphate, scopolamine bromide, decamethonium bromide, mecamylamine hydrochloride, hexamethonium bromide, histamine chloride, mepyramine maleate, promethazine chloride, cirnetidine, methysergide dimaleate. arginine vasopressin, oxytocin,
202
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N . 0. Sjiistrund and E. Klingr All nerves cut
lntacl
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Dec. 6d
CHANGE IN PENILE VOLUME TIME. 2 min
0 PHENTOLAMINE, 14 pmoles/kg
8
-8
0
a
Fig. 2. Spontaneous changes in and effect of phentolamine on penile volume. Blood pressure in this and the other figures is recorded from the left carotid artery. Left panel: Rabbit, 3.0 kg, urethan. Rhythmic changes in penile volume 2 h after severing
of the sympathetic chains, the pelvic and hypogastric nerves. Middle panel: Rabbit, 2.4 kg. urethan. Phentolamine decreases blood pressure and increases penile volume in the “intact” rabbit. Right panel: Rabbit, 4.1 kg, urethan. Sympathetic paravertebral decentralization of the penis 6 days before expt. The pressure drop after phentolamine is moderate. Penile volume is not increased but rather decreased.
isoleucines-angiotensin,lidocaine hydrochloride, sodium pentobarbital and urethan. For manufacturers see Klinge & Sjostrand 1974, 1977a and 6 . Dihydroergotamine mesylate was used in the form of the pharmaceutical product Orstanom@(Sandoz).
Vasoconstrictor innervation
S.
excitutory nerVe.y c?f‘
penile smooth muscles (Figs. 2-6 and Table 1) A. A n u t o w . According to among others Loven (1866). Francois-Franck (1895) and Langley & Anderson (1895) the main part of the vasoconstrictor fibres to the penis come from the lumbosacral symRESULTS AND COMMENTS pathetic chains and reach the penis via the pudic Evidence for myogenic tone and uutomuticity nerves. This was confirmed. Since stimulation of in penile vusculur bed (Fig. 2. See also the pudic nerves rarely produced as forceful reFigs. 3. 6 and 7) sponses as stimulation of the sympathetic chains In vitro studies of various smooth muscle effectors (L&) and since severing of the chains produced a of penile erection indicate a high degree of automa- greater protrusion than cutting of the pudic nerves ticity in most of them (Klinge & Sjostrand 1977~). close to the penis. many fibres must join the pudic This seemed to be the case also in vivo. Rhythmic artery at quite a distance from the penis. Earlier oscillations in penile volume having similar perio- investigators (for ref. see Klinge & Sjostrand 1974) dicity as rhythmic contractions in isolated cavern- have found some vasoconstrictor fibres in the hyous bodies of rabbits were now and then observed pogastric and pelvic nerves. In 2 expts. only we saw in about half of the animals (Fig. 2). These oscilla- faint retractions preceding the usual protrusions tions were not eliminated by section of any of the elicited by stimulation of these nerves. Therefore penile nerves. They could be seen for some minutes these routes seem to be exceptional for constrictor after circulatory arrest in the animal. Thus they fibres in the rabbit. B. Pharmacology. Mecamylamine (30 pmol/kg) might be due to synchronic contractions and relaxations of many smooth muscle fibres in the cavern- suppressed the response to stimulation of the symous bodies rather than to periodicity in the inflow pathetic chains (L&) but not that to stimulation of and/or outflow resistance vessels. However, since the pudic nerves (Fig. 3), i.e. the synaptic relays in removal of the vasoconstrictor tone never resulted the constrictor pathway seem to be located mainly in full penile protrusion (see below) also the inflow in the sacrococcygeal part of the chain (cf. Langley resistance vessels must normally have myogenic & Anderson 1895, 18%). In rabbits with unsevtone. ered vasoconstrictor nerves mecamylamine ( 15-60
Control of penile r~ohrrnr
r Mecamylamine
30 pmoles/ka
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ARTERIAL BLOOD PRESSURE. mm Hg 0 -
TIME. 2 min NPUD SIN STIM 8 sYMP CHAIN STIM
--
. . . . . . . 1 . .
8
0
nz. 8
20 s
28
20
40
54
1 .
nz, 20 s
)tf
Fig. 3. Rabbit. 3.1 kg, urethan, external iliac vessels tied, lateral position. Sympathet-
ic chains cut at LB-LT,electrodes at L7 and on left pudic nerve. Alternate stimulations. Left panel: Control, both stimulations reduce penile volume. Note "rebound" increase in volume after stimulations. Middle panel: 10 min after mecamylamine infusion. The response to stimulation of the sympathetic chains is blocked but not that to pudic nerve stimulation. Right panel: 30 min after ganglionic blockade a slight reappearance of the effect of chain stimulation is noted in the last two stimulations. Pudic nerve stimulations give marked responses. The volume of the penis gradually increased which may partly have been due to accumulation of vasodilating metabolites into the blood as a consequence of the low pressure.
pmol/kg) and hexamethonium (28 pmol/kg) produced clear-cut penile protrusion concomitantly with a lowering of the blood pressure (Fig. 4). Similar protrusions were produced by local infiltration of lidocaine (7.4X M)on the lumbar sympathetic chains or the pudic nerves (Fig. 4). Penile retraction produced by vasoconstrictor stimulation (0.05-
16 Hz)was blocked by guanethidine (6.5-13 pmol/ kg), dihydroergotamine (0.5-2.0 pmol/kg), phenoxybenzamine (1.5 pmol/kg) or phentolamine (2.5-14 pmol/kg). The latter two drugs immediately produced marked drop in blood pressure and clearcut penile protrusion in rabbits with intact constrictor pathways (cf. Fig. 2). Under these circumstan-
A ARTERIAL BLOOD PRESSURE. mm Hg
C
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50
CHANGE IN PENILE VOLUME
TIME, 2 min 20
0
8
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30
I-
EVENT MARKING EVENT
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Lidocaine 7.4 x 1Oe2M on Dudic nerves
NA La. 15 nmolea/kg
Mec.am. I.v. 30 moles/kp
NA i.8. 40 nmoles/kp
Fig. 4. A. Rabbit, 2.1 kg, urethan, ventral position. Infiltration of lidocaine around both
pudic nerves along their dorsal course produces protrusion of the penis. Intra-arterial injection of noradrenaline leads to prompt retraction of the penis. E . Rabbit, 4.0 kg, urethan, dorsal position. Mecamylamine causes pressure drop and penile tumescence. C. In the same rabbit noradrenaline instantly causes detumescence although it considerably rises the blood pressure. Actci
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ARTERIAL BLOOD PRESSURE, mm Hg
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CHANGE IN PENILE VOLUME
0.4
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FREOUENCY
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Fig. 5. Rabbit, 3.8 kg, urethan. Sympathetic chains cut at LrL,, electrodes on L,-S,,
external iliac vessels tied. Responses to stimulation of the chain with varying frequencies. Note that the effect of individual pulses can be traced in the plethysmograrn at 0.05 and 0.1 Hz.Note also biphasic volume decrease during vasoconstrictor stimulation, i.e. and initial rapid phase and a slower second phase. The protrusion after section of the chains was unusually great in this expt.
ces 6.5-1 3 pmol/kg of guanethidine produced only a drop in blood pressure without protrusion of the penis. whereas a transient protrusion was seen after 65 pmol/kg. Dihydroergotamine had but little effect on blood pressure and penile volume, but if the sympathetic chains were cut and the penis protruded this compound retracted the penis. C. Ejykt oj'section of vasoconstrictor pcithwuys. As mentioned above also mechanical interruption of the vasoconstrictor tone produced increase in penile volume. The usual phenomenon was a rapid protrusion followed by a slow (0.5-4 h) or sometimes rapid retraction. In Fig. 6 the initial as well as the final volume range of 10 expts. is illustrated (striped area). As seen, the initial protrusion varied from just slightly above the resting level of the intact penis to about 80% of maximum protrusion produced by stimulation of vasodilator nerves. In most expts. the protrusion produced by cutting the vasoconstrictor pathways was 4 0 4 0 % and the following spontaneous retraction reduced the volume to a level corresponding to the upper limits of the resting volume of the intact penis, i.e. to 20-3076. 0.Response to stirnulation of the syinpnthetic c,lrriiri.s: shripe u i i d frequency-response relationship.
Penes protruded after section of the sympathetic chains responded even to single pulses with a slight retraction and on continuous low frequency stimulation the responses to individual shocks could be traced (Fig. 5 ) . As seen in this figure the response to vasoconstrictor stimulation had two phases: an inA ~ t Pliyskd r Sccoid
/U~J
itial rapid decrease in volume and an additional second slow decrease. This was especially clear within the frequency range of I 4 Hz. Volume decreases in two phases with different slopes have been described on vasoconstrictor stimulation of other organs. The rapid initial phase has been interpreted as due to expulsion of blood from capacitance vessels while the subsequent slow phase has been ascribed to absorption of extravascular fluid caused by relatively more pronounced vasoconstriction in pre- than postcapillary resistance vessels (vide Mellander I%O). Accordingly we consider it likely that the initial rapid phase would chiefly be due to squeezing out of blood from the penis as a consequence of contraction of the smooth muscles in the walls and the trabeculae of the erectile tissue which could be regarded as a kind of capacitance section in the penile bed. The later slower phase, on the other hand, might mainly be due to emptying of the erectile tissue because of greater resistance in the inflow than in the outflow vessels. In Fig. 6 the frequency-response relationship between penile volume and unlimited train stimulation of the sympathetic chains is presented. The plots are based on animals in which cutting of the chains produced 60-80 % initial protrusion. Maximum retraction is obtained with 1-4 Hz.While the plot in Fig. 6 just gives a "static" conception of the effect of vasoconstrictor stimulation the more "dynamic" values of the corresponding emptying rates of the
Control of penile wlrrtnc. PER CENT OF MAXIMUM RETRACTION
205
- OR - PROTRUSION
Fig. 6. Frequency-response relationship between penile volume and stimulation with
"unlimited" pulse train of vasoconstrictor or dilator nerves. 100% Protrusion=O% retraction is the maximum volume obtained by stimulation of vasodilator nerves. 100% Retraction=O% protrusion is the minimum volume obtained by stimulation of vasoconstrictor nerves. All rabbits used for these plots had a mean arterial pressure between 85-1 15 mmHg. Frequency on logarithmic scale. Each point represents mean and range of 3 expts. Dotted area represents range of "resting" volume calculated from 10 expts. The area with most dots indicates the most stationary level. Resting level corresponds to a vasoconstrictor tone in the range of 0.1-0.5 Hz. Striped area indicates volume after section of sympathetic chains: Darker part indicates the initial protrusion, lighter one shows the level after retraction. Calculated from 10 expts. Note the range. For further explanations see text.
penis calculated from the plethysmograms are presented in Table I . The average rate to the peak response as well as the rate during the initial rapid phase of volume decrease are given. The rate of the rapid phase reaches maximum at about 4 Hz while the average rate of volume decrease continues to increase up to 8-16 Hz. This would fit with the above mentioned interpretation of the shape of the response to vasoconstrictor stimulation because functional vasoconstrictor responses of capacitance vessels have maxima at lower frequencies than those of resistance vessels (cf. Mellander 1960, Mellander & Johansson 1968). In this context it should be stressed that the main volume decrease occurred during the initial rapid phase (Fig. 5 ) . E. Estirnrrtion of resting wsotnotor tone l o the pcwis. I n Fig. 6 the results of 10 expts. in which the range of the resting level was calculated against the maximum retraction obtained by vasoconstrictor stimulation and the maximum protrusion obtained by vasodilator stimulation are summarized (dotted area). The dotted area corresponds to vasocon-
strictor stimulation in the frequency range of 0.0s0.5 Hz. The most stationary level (most heavily dotted area) in each expt. corresponds to 0.1-0.2 Hz.That the "normal" vasoconstrictor tone in fact was within this range could be verified in expts. where, after protrusion due to section of the sympathetic chains, the precutting level of penile volume was restored by stimulation with 0.1-0.3 Hz. Thus, provided that there normally is a fairly uniform activation of the penile vasoconstrictor fibres, their vasomotor tone would be of the order of 0.2 Hz. This vasomotor tone is lower than that calculated in similar way for most other vascular beds in anesthetized animals (for ref. see Mellander & Johansson 1968. Folkow & Neil 1971. Oberg 1976). InjlrrtnccJ o f the udrcnrrl tncdirllti irnd yffwts of injwted nor~rclrcncrlitioand ~ t d r ~ ~ n d i n i ~ on penile volume (Fig. 4) i n d r i l l ( i . Cutting or infiltration with lidocaine of splanchnic nerves had no effect on
A. ALIrrnd
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Table I . C d r i h t e d ernpt.vinl: rtites .f'rabbit penis (luring stimulrition with int.riwsing Jrccliri~ric~ii~.t (!f t h r sytnptrthrtii~chains tind corresponditlg fillitlg rtrtes during stiniulrition ofthe pel\-ic*or hypogtistrii. tiiwy1.t Stimulation frequency, Hz 0.05
0. I
A . Average rute to peuk of response Symp. chains 0.I2f0.02 0.17f0.04 Pelv. nerves Hypogastr. n. B. Rutr during rupid phase ($response Symp. chains 0.44f0.13 0.9Sk0.25 Pelv. nerves Hypogastr. n. -
0.2
0.5
I
2
4
0.26f0.07
0.37t0.10 0.1Sf0.04 -
0.53kO. I3 0.13f0.03 -
0.74f0.07 0.13f0.05 0.09t0.06
0.92kO.08
I .74k0.33 0.27f0.04 -
2. I I f0.08 0.29t0.08 -
3.16f0.78 0.5ZfO. 15 0.16fO.l I
4.20k0.71 I .2Ok0.48
-
-
0.98k0. I9
-
0.41f0.05
0.26k0.13
0.55k0.25
Rates in mllmin, meanfS.E., n = 3 . The calculations are based on the same expts. as Fig. 6 and the left panel of Fig. 8. For proper evaluation of the values it should be known that in these expts. the volume difference between maximum protrusion due to vasodilation and maximum retraction due to vasoconstriction was 0.35445 ml. Note that at frequencies below 32 Hz the emptying rates were always faster than the filling rates the difference being more pronounced at lower frequencies and especially during the rapid phase of response. Note also that at frequencies below 8 Hz the filling rates due to stimulation of the pelvic nerves clearly exceeded those due to stimulation of the hypogastric nerves
penile volume in animals with intact or broken penile vasomotor supply. Stimulation (4-32 Hz) of the left splanchnic nerve in animals with intact sympathetic chains produced only faint retractions. However, if the chains were cut and the penis was protruded clear retractions could be obtained with such stimulations. In the most sensitive of the three rabbits in which the right adrenal had been ligated clear retraction was seen with 4 Hz and maximum retraction with 16 Hz (unlimited train stimulation), which corresponded to about 0.2 Hz stimulation of the sympathetic chains. Already stimulation with 4 Hz of the splanchnic nerve had marked effects on the blood pressure. We conclude that adrenal medullary catecholamine secretion hardly participates to any significant degree in normal control of penile volume but could affect it under extreme conditions as e.g. asphyxia (Sjostrand & Klinge 1979).
B. Injec.ticin of norcrdrenrilini~or ridrentilini~. In rabbits with intact sympathetic chains i.a. injections of 10-50 nmol/kg of either of these amines had but faint effects on penile volume. However, if the vasomotor tone had been broken and the penis was protruded prompt retractions were obtained (Fig. 4). I.v. injections of the above amounts of these amines were almost ineffective. In this context it should be mentioned that we never saw any clear protrusion after i.a. injection of adrenaline or noradrenaline, not even after a-adrenoceptor blockade. This also holds true for stimulation of the
splanchnic nerve or the sympathetic chains. Since the adrenal of the rabbit contains almost exclusively adrenaline (Euler 1956) we conclude that we have not obtained any evidence for a significant physiological role of humoral or innervated p-rcceptors in the penile vascular bed, although such are present since terbutaline (2 pmol/kg) produced protrusion in spite of simultaneous fall in blood pressure (cf. Klinge & Sjostrand 1974, 1977li). Vasodilator innervation s. inhibitory nerves (.f penile smooth muscle (Figs. 6-10 and Table I )
A. Anatomy tirid gerieral physiology. Two nerve sets, i.e. the pelvic and the hypogastric nerves, were found to convey erectile fibres. We found no evidence for erectile fibres in the pudic nerve or in the sympathetic chain at the level of L&,. This concept is based on expts. on untreated animals as well as on animals treated with guanethidine. dihydroergotamine, phentolamine or phenoxybenzamine, and is in disagreement with the results of FranGois-Franck (1895) and Bacq ( 1935) on dogs. They found dilator fibres also in these nerves. As expected, the erectile responses were dependent on the arterial blood pressure (Fig. 9). They also were dependent on the phase of rhythmic oscillations if such were present, i.e. the standard stimulation of 20 s had much smaller effect if the penis was spontaneously retracting. Severing of the hypogastric and/or pelvic nerves was without effect on the resting volume of the penis. Thus w e con-
Control of pmile volurne
8
16
32
1 . 1 I fO.I6 0.86f0.40 0.47k0.30
1.25k0.28 I. W0.50 1.0520.76
0.76k0. I5 1.05k0.58 0.67f0.41
4.28k0.80 I .27+0.43 1.33k0.25
4.06f0.93 2.67k 1.40 2.7221.65
2.19f0.73 2.09f 1.21
1.60k0.80
clude that there was no significant tonic discharge in the dilator fibres. B. Pelvic pathway. 1) Pharmacology. The dilator response was blocked by mecamylamine, the doses being in general higher than those required to block the vasomotor tone, i.e. 30-60 pmol/kg. This effect was best seen in animals in which the sympathetic chains had been cut prior to the expt. in order to minimize the decrease of blood pressure (Fig. 7). Thus our stimulation was preganglionic (cf. Langley & Anderson 1895). We did not observe any
ARTERIAL BLOOD PRESSURE, rnm Hg
:,j 0
overt effect of even very large doses of atropine (140 pmol/kg) or scopolamine (250 pmol/kg) in atropine sensitive or semiresistant rabbits on the erectile response to pelvic nerve stimulation (20 s, 05-16 Hz). This also applies to propranolol (10 pmol/kg), mepyramine (25 pmol/kg), cimetidine (40 pmol/kg) and methysergide (4.3 pmoVkg). Furthermore, the dilator response of the selectively perfused penile artery to pelvic nerve stimulation (6-12 Hz,30 s. 2 expts.) was not overtly affected even by long time perfusion with 2 . 6 ~ M scopolamine (Fig. IOc). This concentration of scopolamine blocked the effect of acetylcholine injected into the penile artery (Fig. 10h). 2) Frequency-response relationship. Threshold frequency for a response was 0.1-0.5 Hz.With 0.5 Hz steady and reproducible effects were obtained in most expts. Fig. 6 shows a frequency-response plot to unlimited stimulation. The expts. were performed on rabbits with intact vasomotor tone. Maximum protrusion is obtained within 1-2 Hz.While it is of interest for the understanding of long lasting erections as may be the case e.g. in man, the curve gives, however, but little of the “dynamics” of erection. The corresponding filling rates are therefore presented in Table I in which the calculated average as well as maximum rates are given. The erectile responses were not distinctly biphasic as were the retractile responses. In general there was a lag phase, a short slow phase and a rapid phase which eventually levelled off. At increasing fre-
Syrnp
chain cut 3 d before exp
_ 1
CHANGE IN PENILE VOLUME h
TIME
2 min
, . . . . . 12,
0 N PELV STlM 8 Hz 20 s
N H Y P STlM 8 Hz 20 s MECAMYLAMINE 60 prnolesh
207
-
I
58
.
.
.
.
,
68
82
94
F
L
Fig. 7. Rabbit, 4.1 kg, urethane, sympathetic chains cut 3 days prior to expt. Left panel: Responses to pelvic and hypogastric nerve stimulation. Note size and duration of responses and also rapid phase of retraction. Middle panel: Mecarnylamine is without significant effect on penile volume (cf. Fig. 4 8 ) . Right panel: After ganglionic blockade the responses to hypogastric and pelvic nerve stimulation are severely depressed. Note also that ganglionic blockade has only a moderate effect on blood pressure.
N . 0 . Sjiistrund atid E . Klitigc.
208
Per cent of max response
Time 10 peak 01 response 51
Principal mechanisms controlling penile retraction and protrusion in rabbits NILS 0. SJOSTRANDand ERIK KLINGE Department of Physiology, Karolinska Institutet, Stockholm, Sweden, and the Department of Pharmacology, University of Helsinki, Finland
SJOSTRAND, N. 0. & KLINGE, E.: Principal mechanisms controlling penile retraction and protrusion in rabbits. Acta Physiol Scand 1979, 106: 199-214. Received 30 Nov. 1978. ISSN 00016772. Dept. of Physiology, Karolinska Inst. and Dept. of Pharmacology, University of Helsinki, Finland. The effects on penile volume of nerve stimulations and drugs injected into the systemic circulation were studied plethysmographically. Dilator responses at selective perfusion of the penile artery were studied by measuring the perfusion pressure. The main results and conclusions are: The penis has an adrenergic vasoconstrictor supply coming from the sacrococcygeal parts of the sympathetic chains. A very low (0.2 Hz) vasomotor tone keeps the penis relaxed. If this tone is interrupted the penis will protrude but autoregulation will soon take over and eventually produce hyperinvolution of the penis. Two vasodilator paths, both with pelvic ganglionic relays, were found. I ) The pelvic parasympathetic nerves, probably having mainly non-cholinergic postganglionic neurons and operating quite effectively at low frequencies. 2) The sympathetic hypogastric nerves, presumably having at least partly cholinergic postganglionic neurons which, apart from muscarinic dilation of minute inflow resistance vessels to the erectile tissue, may also work by suppression of excitatory adrenergic neurotransmission. The pelvic and hypogastric vasodilator outflows work synergistically. The vasoconstrictor nerves are very strong and efficient antagonists of the vasodilator nerves. Key words: Penis, erection, autonomic innervation, vasodilation, smooth muscle
A primary event in mammalian penile erection is dilation of the arterial branches forming the “inflow resistance” vessels to the cavernous bodies. This leads to a situation where the “outflow resistance” exceeds the inflow resistance and consequently the erectile tissue will be engorged. Conversely, vasoconstriction forms the basis of the flaccid state of the penis. The nature of penile vasodilation has been a matter of dispute for a long time (vide Klinge & Sjostrand 1974). Two main issues have been debated. 1) Are the pelvic parasympathetic nerves, i.e. the nervi erigentes of Eckhard (1863), the only path for penile vasodilator fibres? This seems to be the predominant view apparently based on the works of Langley & Anderson (1895) and Semans & Langworthy (1938) where other routes are refuted. However, there are continental reports indicating that erector fibres also could travel along the sympathetic system, especially the hypogastric nerves (Eckhard 1876, FranGois-Franck 1895, Bacq 1935,
Bessou & Laporte 1961, Thielen, Renders & Rectern 1969). 2) Are the dilator fibres cholinergic, as generally assumed, or not? In fact, this question is much older than the concept of cholinergic nerves and goes back to a dispute on whether atropine “paralyzes” (Nikolsky 1879) the nervi erigentes or not (Anrep & Cybulski 1884). In this century a cholinergic transmission of erection has been proposed by e.g. Henderson & Roepke (1933), Bacq (1935). Oppenheimer (19381, Bessou & Laporte (1961) and Hukovic & Bubic (1%7), while it has been doubted by e.g. Luduena & Grigds (1966) and Dorr & Brody (1967). We have criticized the pharmacological evidence presented by earlier investigators, the main objections being the combined use of preganglionic stimulation and very high frequencies which make it difficult to evaluate the point of attack and the effect of drugs such as atropine and physostigmine (Klinge & Sjostrand 1974). Concerning the putative sympathetic erector fibres there is also an objection Actti
Pli.vsiol Srand 106
200
N . 0.Sjiistrund
(itid
E . Klingr
against the use of a-adrenergic blocking ergot compounds (Bacq 1935, Bessou & Laporte 1961) in order to make the vasodilation clear, because the observed effect could be d u e t o adrenergic p-receptors. Experiments on various isolated smooth muscle effectors of penile erection have led us to the conclusion that neurogenic relaxation of these smooth muscles is hardly cholinergic although cholinergic nerves may contribute t o erection by prejunctional inhibition of excitatory adrenergic neurotransmission to these effectors (Klinge & Sjostrand 1974, 1977ri and b ) . In this report we present results from in vivo studies performed with the aim to obtain information on the basic myogenic and neurogenic control mechanisms of penile volume. The rabbit, having a penis rather similar to that of man (Fujimoto & Takeshige 197% was chosen as experimental animal. A preliminary account of the results has been presented (Sjostrand & Klinge 1978).
MATERIAL A N D M E T H ODS Animuls
This and the subsequent report (Sjostrand & Klinge 1979) are based on expts. on 58 rabbits of different breeds; 32 were albinos of the New Zealand race or native strains. B.wt. was in the range of 2.0-5.5 kg. Operrrtiw procedures
I . Generul prepurution und care. The rabbits were starved for 12-18 h and anesthetized with urethan (16-20 mmol/kg i.v.). They were tracheostomized and a T-tube was inserted. The abdomen was opened with a midline incision. The inferior and superior mesenteric arteries and the corresponding branches of the portal vein were tied and the gut was removed between the duodenum and the oral end of the rectum. The ureters were cannulated and the bladder was emptied. The vessels of the bladder were tied and the bladder was resected above the trigonum area. A cotton-wool pellet soaked in vaseline was introduced into the urethra through the opened bladder and placed at the level of the pubic bone in order to prevent secretion from the male sex accessories into the penis. The vasa deferentia were ligated in the ampullary region and the seminal vesicle was slit in its tips and the secretion was removed. A catheter for i.v. infusions was introduced into either a femoral or an external jugular vein. In some expts. a catheter for i.a. injections was introduced into a femoral or a subclavian artery and pushed to the aortic origin of the vessel. Arterial blood pressure was recorded from one of the carotid arteries (usually the left) or a femoral artery. I n expts. in which the sympathetic chains were cut and stimulated the external iliac vessels were usually tied in order to prevent drop of blood pressure and pooling of blood in the hind legs. In some expts. artificial Avfrr Ph.wio/ Sc~rnd/IN
ventilation was given under decarnethonium ( 1.1-2.4 pmol/kg i.v.) blockade. After evisceration the animals received 10 ml 6 9i dextran in saline (Macrodex@,Pharmacia) per kg b.wt. During the course of the expt. the rabbits received 2-6 rnl of each of the following solutions per h: glucose-fructohr (both 0.56 M), NaHCO, 1.6x lo-' M. 6 % dextran and Ringer or saline. 2. Plethysmogrriphy ofthe penis. The attachment of the penile skin to the rectum was severed. The opening in the penile skin as well as that in the rectal skin were firmly closed by sutures. The preputium was elevated and its inner layer was separated from the penis by a circular incision through the attachment beneath the glans penib. Via this incision the entire penis was stripped by blunt dissection. The skin tube covering the penis was left in continuity with the pubic and the perineal skin. The skinned penis was lubricated with paraffin oil and a greased glass plethysmograph provided with 3 hooks was introduced into the skin tube and placed around the mentulum. The plethysmograph was resting on the dome formed by the perineal striated muscles encircling the crura and the bulb of the penis. Ligatures from the penile skin to the hooks kept the plethysmograph in place and complete sealing was achieved by tying threads around the base of the penile skin and plethysmograph (Fig. I ) . The recording system was tilled with 7-8 ml of air and care was taken to keep the room temperature constant. 3. Selective perfusion of the penile urtery. The penis. the pubic, perineal and sacrococcygeal regions as well as the hips were flayed. Via blunt dissection in the genitofemoral groove, cutting off covering muscles and removing the tuber ossis ischii with its muscular insertions the internal pudic artery was identified in "Alcock's canal". In the 5 New Zealand rabbits used in this type of expt. there was quite a difference in size of the penile arteries on the two sides. The smaller artery was ligated while the larger was cannulated at its origin, i.e. where the terminal pudic artery divides into the penile and rectal branches. All visible penile veins were ligated centrally and cut peripherally and the tip of the glans penis was cut off to permit outflow of perfusion fluid. The penile artery was perfused with Tyrode solution (Klinge & Sjostrand 1974) which contained 2 % FicolP (Pharmacia), M.w. about 77000. and 1.5% FicolP, M.w. about 47000, as colloid and was aerated with 6S%, CO, in 0, and kept at about 34°C. Perfusion rate was 1.75-3.0 ml/min and histamine (0.S5.0x lo-' M) was added to induce sufficient vascular tone. i.e. an effective initial perfusion pressure close to 100 mmHg. The pressure was measured via a T-tube inserted into the arterial cannula. An inlet for injections was placed between the reservoir and the pump close to the latter. 4 . Dissection and stimulation of nerves. Good descriptions of the pelvic, hypogastric and pudic nerves in the rabbit and of the approach to them are given by Langley & Anderson (1895, 18%). These guidelines were essentially followed. The lumbosacral sympathetic chains were identified and prepared at the level of LBS1. The left splanchnic nerve was isolated rather close to the adrenal. In three expts. in which adrenomedullary stimulation was performed the right adrenal was separated from the circulation by tight ligatures. The vagus nerves were isolated a1
SKIN
PLETHYSMOGRAPH PENIS
LIGATURES
Fig. 1 . Schematic drawing of the glass plethysmograph in
situ. Frontal view. The plethysmograph is connected via a low pressure transducer to a Grass polygraph. The degree of protrusion of the rabbit penis, which is retracted in the above illustration, is also directly visible. For further explanations see "Material and Methods".
about the level of the thyroid cartilago. The cut nerves were placed on hook-shaped platinum electrodes ( 2 mm apart) shielded underneath with plexiglass and provided on the upper side with a movable plexiglass rod for fixing the nerve, All electrodes were provided with holders designed specially for each nerve. The holders were fixed to the operation table in order to keep the electrodes in position during the expt. Paraffin oil in ample amount was used for isolation. The nerves were stimulated with square wave pulses (4-6 ms) and "supramaximal" voltage (15-25 V). Unless otherwise stated, all stimulations were bilateral. The usual stimulation period was 20 s . This will be referred to as standard stimulation. In some frequency-response expts. we stimulated until the response became stabilized at a fixed level. This will be referred to as stimulation with unlimited train. 5. Section of lutnbosacrul sympathetic chains prior to expts. Six rabbits were pretreated with promethazine (16 pmol/kg i.m.) and anesthetized with sodium pentobarbital (120-200 pmol/kg i.v.). The abdomen was opened along the linea alba and the paravertebral chains were dissected free around the ganglia at L,. The cranial and caudal connections of these ganglia and those at L,, and S, were removed. whereafter the abdomen was closed. The animals were used for expts. 2. 3, 4, 6, 14 or 21 d after operation. Special considerations in the unalysiu of the effects of antirnuscarinic drugs on erectile responses
The use of antimuscarinic drugs as atropine or scopolamine in in vivo expts. on rabbits is encumbered with many drawbacks. Many rabbits have an inherited atropinesterase in plasma, liver and several other organs, which rapidly inactivates both atropine and scopolamine (for ref. see e.g. Kalow 1%2, Cauthen et al. 1976). There further is the problem of reaching a sufficient concentration of drugs in a normally poorly perfused organ as the penis. Therefore all effects of atropine and scopolamine on the response of the penis to pelvic or hypogastric nerve stimulation were evaluated against the effect of the drugs on the response of the heart to stimulation on the right vagus nerve (20 s , 15-25 Hz). This response is one of the most atropine
sensitive cholinergic nerve effects (e.g. Ursillo 1961). Consequently only in expts. where this effect could be blocked one might obtain significant pharmacological data on the possible muscarinic nature of the erectile response. We could divide our rabbits into 3 groups: a small group (only 3 rabbits) in which the vagal bradycardia could be blocked for several h by moderate doses (4-20 pmol/kg) of the drugs, a main group in which just a short-lived (3-15 min) suppression of the bradycardia was obtained with even huge doses (70-170 pmol/kg) of the alkaloids, and finally one group where even grotesque doses (more than 216 pmol/kg) were without any effect. These groups were defined as atropine sensitive. semiresistant and resistant, respectively. The difficulties in evaluating the effects of the antimuscarinic drugs prompted us to do the perfusion expts. Atropine and scopolamine also have ganglionic blocking properties (vide Klinge & Sjostrand 1974) which could impair the analysis. This possibility was checked by recording contractions of the seminal vesicle to injected noradrenaline and to hypogastric nerve stimulation which is preganglionic to that organ (vide Sjostrand 1965). We obtained no clear-cut evidence for a ganglionic action of the alkaloids when doses of 4-40 pmol/kg were used. However. with the higher doses. which we had to use in the perfusion expts. and also in the expts. on atropine sensitive rabbits, an interference of a ganglionic or postganglionic nicotinic point of attack cannut be excluded. Equipment Recordings were made on a Grass polygraph. Statham transducers were used for pressure recordings and a Grass volumetric low-pressure transducer (PT5) was used in plethysmography. A Sigma motor pump was used in the perfusion expts. Grass S4, S44, S5 or SD 9 stimulators delivered the shocks. Slow infusions were made with a Braun infusion pump. Temperatures were measured with Gold Brand precision thermometers and a Harvard respirator was used for artificial ventilation. Some critical comments on the tnethodologv
During evisceration the rectal temperature fell to 35-33°C and stayed at this level throughout the expt. despite warming. Together with the damping of the recording system this means that our quantitative data are sluggish compared to those of "real life". The airfilled system in plethysmography had of course the errors of this method. Nervous injuries due to preparation should not be overlooked. Especially the pelvic nerves were difficult to prepare satisfactorily in contrast to the easy hypogastric nerves. Drugs The following pure salts, bases and peptides were used: I-noradrenaline~-bitartrate, I-adrenaline-d-bitartrate, terbutaline sulphate, phenoxybenzamine hydrochloride, phentolamine methane sulphonate, propranolol hydrochloride, guanethidine sulphate, acetylcholine chloride, neostigmine bromide, atropine sulphate, scopolamine bromide, decamethonium bromide, mecamylamine hydrochloride, hexamethonium bromide, histamine chloride, mepyramine maleate, promethazine chloride, cirnetidine, methysergide dimaleate. arginine vasopressin, oxytocin,
202
-
N . 0. Sjiistrund and E. Klingr All nerves cut
lntacl
-
0
Dec. 6d
CHANGE IN PENILE VOLUME TIME. 2 min
0 PHENTOLAMINE, 14 pmoles/kg
8
-8
0
a
Fig. 2. Spontaneous changes in and effect of phentolamine on penile volume. Blood pressure in this and the other figures is recorded from the left carotid artery. Left panel: Rabbit, 3.0 kg, urethan. Rhythmic changes in penile volume 2 h after severing
of the sympathetic chains, the pelvic and hypogastric nerves. Middle panel: Rabbit, 2.4 kg. urethan. Phentolamine decreases blood pressure and increases penile volume in the “intact” rabbit. Right panel: Rabbit, 4.1 kg, urethan. Sympathetic paravertebral decentralization of the penis 6 days before expt. The pressure drop after phentolamine is moderate. Penile volume is not increased but rather decreased.
isoleucines-angiotensin,lidocaine hydrochloride, sodium pentobarbital and urethan. For manufacturers see Klinge & Sjostrand 1974, 1977a and 6 . Dihydroergotamine mesylate was used in the form of the pharmaceutical product Orstanom@(Sandoz).
Vasoconstrictor innervation
S.
excitutory nerVe.y c?f‘
penile smooth muscles (Figs. 2-6 and Table 1) A. A n u t o w . According to among others Loven (1866). Francois-Franck (1895) and Langley & Anderson (1895) the main part of the vasoconstrictor fibres to the penis come from the lumbosacral symRESULTS AND COMMENTS pathetic chains and reach the penis via the pudic Evidence for myogenic tone and uutomuticity nerves. This was confirmed. Since stimulation of in penile vusculur bed (Fig. 2. See also the pudic nerves rarely produced as forceful reFigs. 3. 6 and 7) sponses as stimulation of the sympathetic chains In vitro studies of various smooth muscle effectors (L&) and since severing of the chains produced a of penile erection indicate a high degree of automa- greater protrusion than cutting of the pudic nerves ticity in most of them (Klinge & Sjostrand 1977~). close to the penis. many fibres must join the pudic This seemed to be the case also in vivo. Rhythmic artery at quite a distance from the penis. Earlier oscillations in penile volume having similar perio- investigators (for ref. see Klinge & Sjostrand 1974) dicity as rhythmic contractions in isolated cavern- have found some vasoconstrictor fibres in the hyous bodies of rabbits were now and then observed pogastric and pelvic nerves. In 2 expts. only we saw in about half of the animals (Fig. 2). These oscilla- faint retractions preceding the usual protrusions tions were not eliminated by section of any of the elicited by stimulation of these nerves. Therefore penile nerves. They could be seen for some minutes these routes seem to be exceptional for constrictor after circulatory arrest in the animal. Thus they fibres in the rabbit. B. Pharmacology. Mecamylamine (30 pmol/kg) might be due to synchronic contractions and relaxations of many smooth muscle fibres in the cavern- suppressed the response to stimulation of the symous bodies rather than to periodicity in the inflow pathetic chains (L&) but not that to stimulation of and/or outflow resistance vessels. However, since the pudic nerves (Fig. 3), i.e. the synaptic relays in removal of the vasoconstrictor tone never resulted the constrictor pathway seem to be located mainly in full penile protrusion (see below) also the inflow in the sacrococcygeal part of the chain (cf. Langley resistance vessels must normally have myogenic & Anderson 1895, 18%). In rabbits with unsevtone. ered vasoconstrictor nerves mecamylamine ( 15-60
Control of penile r~ohrrnr
r Mecamylamine
30 pmoles/ka
203
1
ARTERIAL BLOOD PRESSURE. mm Hg 0 -
TIME. 2 min NPUD SIN STIM 8 sYMP CHAIN STIM
--
. . . . . . . 1 . .
8
0
nz. 8
20 s
28
20
40
54
1 .
nz, 20 s
)tf
Fig. 3. Rabbit. 3.1 kg, urethan, external iliac vessels tied, lateral position. Sympathet-
ic chains cut at LB-LT,electrodes at L7 and on left pudic nerve. Alternate stimulations. Left panel: Control, both stimulations reduce penile volume. Note "rebound" increase in volume after stimulations. Middle panel: 10 min after mecamylamine infusion. The response to stimulation of the sympathetic chains is blocked but not that to pudic nerve stimulation. Right panel: 30 min after ganglionic blockade a slight reappearance of the effect of chain stimulation is noted in the last two stimulations. Pudic nerve stimulations give marked responses. The volume of the penis gradually increased which may partly have been due to accumulation of vasodilating metabolites into the blood as a consequence of the low pressure.
pmol/kg) and hexamethonium (28 pmol/kg) produced clear-cut penile protrusion concomitantly with a lowering of the blood pressure (Fig. 4). Similar protrusions were produced by local infiltration of lidocaine (7.4X M)on the lumbar sympathetic chains or the pudic nerves (Fig. 4). Penile retraction produced by vasoconstrictor stimulation (0.05-
16 Hz)was blocked by guanethidine (6.5-13 pmol/ kg), dihydroergotamine (0.5-2.0 pmol/kg), phenoxybenzamine (1.5 pmol/kg) or phentolamine (2.5-14 pmol/kg). The latter two drugs immediately produced marked drop in blood pressure and clearcut penile protrusion in rabbits with intact constrictor pathways (cf. Fig. 2). Under these circumstan-
A ARTERIAL BLOOD PRESSURE. mm Hg
C
B
1
'""1 7 150
50
CHANGE IN PENILE VOLUME
TIME, 2 min 20
0
8
24
30
I-
EVENT MARKING EVENT
...._....
0
Lidocaine 7.4 x 1Oe2M on Dudic nerves
NA La. 15 nmolea/kg
Mec.am. I.v. 30 moles/kp
NA i.8. 40 nmoles/kp
Fig. 4. A. Rabbit, 2.1 kg, urethan, ventral position. Infiltration of lidocaine around both
pudic nerves along their dorsal course produces protrusion of the penis. Intra-arterial injection of noradrenaline leads to prompt retraction of the penis. E . Rabbit, 4.0 kg, urethan, dorsal position. Mecamylamine causes pressure drop and penile tumescence. C. In the same rabbit noradrenaline instantly causes detumescence although it considerably rises the blood pressure. Actci
Pliysiol Sccincl I 0 6
204
N . 0 . Sjiistrand rind E . Klirige
ARTERIAL BLOOD PRESSURE, mm Hg
:I
-
-r
50
CHANGE IN PENILE VOLUME
0.4
ml
TIME, 2 min
-
SYMP. CHAIN STIM.
FREOUENCY
0.05
01
0.2
05
1
2
HZ
Fig. 5. Rabbit, 3.8 kg, urethan. Sympathetic chains cut at LrL,, electrodes on L,-S,,
external iliac vessels tied. Responses to stimulation of the chain with varying frequencies. Note that the effect of individual pulses can be traced in the plethysmograrn at 0.05 and 0.1 Hz.Note also biphasic volume decrease during vasoconstrictor stimulation, i.e. and initial rapid phase and a slower second phase. The protrusion after section of the chains was unusually great in this expt.
ces 6.5-1 3 pmol/kg of guanethidine produced only a drop in blood pressure without protrusion of the penis. whereas a transient protrusion was seen after 65 pmol/kg. Dihydroergotamine had but little effect on blood pressure and penile volume, but if the sympathetic chains were cut and the penis protruded this compound retracted the penis. C. Ejykt oj'section of vasoconstrictor pcithwuys. As mentioned above also mechanical interruption of the vasoconstrictor tone produced increase in penile volume. The usual phenomenon was a rapid protrusion followed by a slow (0.5-4 h) or sometimes rapid retraction. In Fig. 6 the initial as well as the final volume range of 10 expts. is illustrated (striped area). As seen, the initial protrusion varied from just slightly above the resting level of the intact penis to about 80% of maximum protrusion produced by stimulation of vasodilator nerves. In most expts. the protrusion produced by cutting the vasoconstrictor pathways was 4 0 4 0 % and the following spontaneous retraction reduced the volume to a level corresponding to the upper limits of the resting volume of the intact penis, i.e. to 20-3076. 0.Response to stirnulation of the syinpnthetic c,lrriiri.s: shripe u i i d frequency-response relationship.
Penes protruded after section of the sympathetic chains responded even to single pulses with a slight retraction and on continuous low frequency stimulation the responses to individual shocks could be traced (Fig. 5 ) . As seen in this figure the response to vasoconstrictor stimulation had two phases: an inA ~ t Pliyskd r Sccoid
/U~J
itial rapid decrease in volume and an additional second slow decrease. This was especially clear within the frequency range of I 4 Hz. Volume decreases in two phases with different slopes have been described on vasoconstrictor stimulation of other organs. The rapid initial phase has been interpreted as due to expulsion of blood from capacitance vessels while the subsequent slow phase has been ascribed to absorption of extravascular fluid caused by relatively more pronounced vasoconstriction in pre- than postcapillary resistance vessels (vide Mellander I%O). Accordingly we consider it likely that the initial rapid phase would chiefly be due to squeezing out of blood from the penis as a consequence of contraction of the smooth muscles in the walls and the trabeculae of the erectile tissue which could be regarded as a kind of capacitance section in the penile bed. The later slower phase, on the other hand, might mainly be due to emptying of the erectile tissue because of greater resistance in the inflow than in the outflow vessels. In Fig. 6 the frequency-response relationship between penile volume and unlimited train stimulation of the sympathetic chains is presented. The plots are based on animals in which cutting of the chains produced 60-80 % initial protrusion. Maximum retraction is obtained with 1-4 Hz.While the plot in Fig. 6 just gives a "static" conception of the effect of vasoconstrictor stimulation the more "dynamic" values of the corresponding emptying rates of the
Control of penile wlrrtnc. PER CENT OF MAXIMUM RETRACTION
205
- OR - PROTRUSION
Fig. 6. Frequency-response relationship between penile volume and stimulation with
"unlimited" pulse train of vasoconstrictor or dilator nerves. 100% Protrusion=O% retraction is the maximum volume obtained by stimulation of vasodilator nerves. 100% Retraction=O% protrusion is the minimum volume obtained by stimulation of vasoconstrictor nerves. All rabbits used for these plots had a mean arterial pressure between 85-1 15 mmHg. Frequency on logarithmic scale. Each point represents mean and range of 3 expts. Dotted area represents range of "resting" volume calculated from 10 expts. The area with most dots indicates the most stationary level. Resting level corresponds to a vasoconstrictor tone in the range of 0.1-0.5 Hz. Striped area indicates volume after section of sympathetic chains: Darker part indicates the initial protrusion, lighter one shows the level after retraction. Calculated from 10 expts. Note the range. For further explanations see text.
penis calculated from the plethysmograms are presented in Table I . The average rate to the peak response as well as the rate during the initial rapid phase of volume decrease are given. The rate of the rapid phase reaches maximum at about 4 Hz while the average rate of volume decrease continues to increase up to 8-16 Hz. This would fit with the above mentioned interpretation of the shape of the response to vasoconstrictor stimulation because functional vasoconstrictor responses of capacitance vessels have maxima at lower frequencies than those of resistance vessels (cf. Mellander 1960, Mellander & Johansson 1968). In this context it should be stressed that the main volume decrease occurred during the initial rapid phase (Fig. 5 ) . E. Estirnrrtion of resting wsotnotor tone l o the pcwis. I n Fig. 6 the results of 10 expts. in which the range of the resting level was calculated against the maximum retraction obtained by vasoconstrictor stimulation and the maximum protrusion obtained by vasodilator stimulation are summarized (dotted area). The dotted area corresponds to vasocon-
strictor stimulation in the frequency range of 0.0s0.5 Hz. The most stationary level (most heavily dotted area) in each expt. corresponds to 0.1-0.2 Hz.That the "normal" vasoconstrictor tone in fact was within this range could be verified in expts. where, after protrusion due to section of the sympathetic chains, the precutting level of penile volume was restored by stimulation with 0.1-0.3 Hz. Thus, provided that there normally is a fairly uniform activation of the penile vasoconstrictor fibres, their vasomotor tone would be of the order of 0.2 Hz. This vasomotor tone is lower than that calculated in similar way for most other vascular beds in anesthetized animals (for ref. see Mellander & Johansson 1968. Folkow & Neil 1971. Oberg 1976). InjlrrtnccJ o f the udrcnrrl tncdirllti irnd yffwts of injwted nor~rclrcncrlitioand ~ t d r ~ ~ n d i n i ~ on penile volume (Fig. 4) i n d r i l l ( i . Cutting or infiltration with lidocaine of splanchnic nerves had no effect on
A. ALIrrnd
206
N . 0. Sjijstrritid and E . Klinge
Table I . C d r i h t e d ernpt.vinl: rtites .f'rabbit penis (luring stimulrition with int.riwsing Jrccliri~ric~ii~.t (!f t h r sytnptrthrtii~chains tind corresponditlg fillitlg rtrtes during stiniulrition ofthe pel\-ic*or hypogtistrii. tiiwy1.t Stimulation frequency, Hz 0.05
0. I
A . Average rute to peuk of response Symp. chains 0.I2f0.02 0.17f0.04 Pelv. nerves Hypogastr. n. B. Rutr during rupid phase ($response Symp. chains 0.44f0.13 0.9Sk0.25 Pelv. nerves Hypogastr. n. -
0.2
0.5
I
2
4
0.26f0.07
0.37t0.10 0.1Sf0.04 -
0.53kO. I3 0.13f0.03 -
0.74f0.07 0.13f0.05 0.09t0.06
0.92kO.08
I .74k0.33 0.27f0.04 -
2. I I f0.08 0.29t0.08 -
3.16f0.78 0.5ZfO. 15 0.16fO.l I
4.20k0.71 I .2Ok0.48
-
-
0.98k0. I9
-
0.41f0.05
0.26k0.13
0.55k0.25
Rates in mllmin, meanfS.E., n = 3 . The calculations are based on the same expts. as Fig. 6 and the left panel of Fig. 8. For proper evaluation of the values it should be known that in these expts. the volume difference between maximum protrusion due to vasodilation and maximum retraction due to vasoconstriction was 0.35445 ml. Note that at frequencies below 32 Hz the emptying rates were always faster than the filling rates the difference being more pronounced at lower frequencies and especially during the rapid phase of response. Note also that at frequencies below 8 Hz the filling rates due to stimulation of the pelvic nerves clearly exceeded those due to stimulation of the hypogastric nerves
penile volume in animals with intact or broken penile vasomotor supply. Stimulation (4-32 Hz) of the left splanchnic nerve in animals with intact sympathetic chains produced only faint retractions. However, if the chains were cut and the penis was protruded clear retractions could be obtained with such stimulations. In the most sensitive of the three rabbits in which the right adrenal had been ligated clear retraction was seen with 4 Hz and maximum retraction with 16 Hz (unlimited train stimulation), which corresponded to about 0.2 Hz stimulation of the sympathetic chains. Already stimulation with 4 Hz of the splanchnic nerve had marked effects on the blood pressure. We conclude that adrenal medullary catecholamine secretion hardly participates to any significant degree in normal control of penile volume but could affect it under extreme conditions as e.g. asphyxia (Sjostrand & Klinge 1979).
B. Injec.ticin of norcrdrenrilini~or ridrentilini~. In rabbits with intact sympathetic chains i.a. injections of 10-50 nmol/kg of either of these amines had but faint effects on penile volume. However, if the vasomotor tone had been broken and the penis was protruded prompt retractions were obtained (Fig. 4). I.v. injections of the above amounts of these amines were almost ineffective. In this context it should be mentioned that we never saw any clear protrusion after i.a. injection of adrenaline or noradrenaline, not even after a-adrenoceptor blockade. This also holds true for stimulation of the
splanchnic nerve or the sympathetic chains. Since the adrenal of the rabbit contains almost exclusively adrenaline (Euler 1956) we conclude that we have not obtained any evidence for a significant physiological role of humoral or innervated p-rcceptors in the penile vascular bed, although such are present since terbutaline (2 pmol/kg) produced protrusion in spite of simultaneous fall in blood pressure (cf. Klinge & Sjostrand 1974, 1977li). Vasodilator innervation s. inhibitory nerves (.f penile smooth muscle (Figs. 6-10 and Table I )
A. Anatomy tirid gerieral physiology. Two nerve sets, i.e. the pelvic and the hypogastric nerves, were found to convey erectile fibres. We found no evidence for erectile fibres in the pudic nerve or in the sympathetic chain at the level of L&,. This concept is based on expts. on untreated animals as well as on animals treated with guanethidine. dihydroergotamine, phentolamine or phenoxybenzamine, and is in disagreement with the results of FranGois-Franck (1895) and Bacq ( 1935) on dogs. They found dilator fibres also in these nerves. As expected, the erectile responses were dependent on the arterial blood pressure (Fig. 9). They also were dependent on the phase of rhythmic oscillations if such were present, i.e. the standard stimulation of 20 s had much smaller effect if the penis was spontaneously retracting. Severing of the hypogastric and/or pelvic nerves was without effect on the resting volume of the penis. Thus w e con-
Control of pmile volurne
8
16
32
1 . 1 I fO.I6 0.86f0.40 0.47k0.30
1.25k0.28 I. W0.50 1.0520.76
0.76k0. I5 1.05k0.58 0.67f0.41
4.28k0.80 I .27+0.43 1.33k0.25
4.06f0.93 2.67k 1.40 2.7221.65
2.19f0.73 2.09f 1.21
1.60k0.80
clude that there was no significant tonic discharge in the dilator fibres. B. Pelvic pathway. 1) Pharmacology. The dilator response was blocked by mecamylamine, the doses being in general higher than those required to block the vasomotor tone, i.e. 30-60 pmol/kg. This effect was best seen in animals in which the sympathetic chains had been cut prior to the expt. in order to minimize the decrease of blood pressure (Fig. 7). Thus our stimulation was preganglionic (cf. Langley & Anderson 1895). We did not observe any
ARTERIAL BLOOD PRESSURE, rnm Hg
:,j 0
overt effect of even very large doses of atropine (140 pmol/kg) or scopolamine (250 pmol/kg) in atropine sensitive or semiresistant rabbits on the erectile response to pelvic nerve stimulation (20 s, 05-16 Hz). This also applies to propranolol (10 pmol/kg), mepyramine (25 pmol/kg), cimetidine (40 pmol/kg) and methysergide (4.3 pmoVkg). Furthermore, the dilator response of the selectively perfused penile artery to pelvic nerve stimulation (6-12 Hz,30 s. 2 expts.) was not overtly affected even by long time perfusion with 2 . 6 ~ M scopolamine (Fig. IOc). This concentration of scopolamine blocked the effect of acetylcholine injected into the penile artery (Fig. 10h). 2) Frequency-response relationship. Threshold frequency for a response was 0.1-0.5 Hz.With 0.5 Hz steady and reproducible effects were obtained in most expts. Fig. 6 shows a frequency-response plot to unlimited stimulation. The expts. were performed on rabbits with intact vasomotor tone. Maximum protrusion is obtained within 1-2 Hz.While it is of interest for the understanding of long lasting erections as may be the case e.g. in man, the curve gives, however, but little of the “dynamics” of erection. The corresponding filling rates are therefore presented in Table I in which the calculated average as well as maximum rates are given. The erectile responses were not distinctly biphasic as were the retractile responses. In general there was a lag phase, a short slow phase and a rapid phase which eventually levelled off. At increasing fre-
Syrnp
chain cut 3 d before exp
_ 1
CHANGE IN PENILE VOLUME h
TIME
2 min
, . . . . . 12,
0 N PELV STlM 8 Hz 20 s
N H Y P STlM 8 Hz 20 s MECAMYLAMINE 60 prnolesh
207
-
I
58
.
.
.
.
,
68
82
94
F
L
Fig. 7. Rabbit, 4.1 kg, urethane, sympathetic chains cut 3 days prior to expt. Left panel: Responses to pelvic and hypogastric nerve stimulation. Note size and duration of responses and also rapid phase of retraction. Middle panel: Mecarnylamine is without significant effect on penile volume (cf. Fig. 4 8 ) . Right panel: After ganglionic blockade the responses to hypogastric and pelvic nerve stimulation are severely depressed. Note also that ganglionic blockade has only a moderate effect on blood pressure.
N . 0 . Sjiistrund atid E . Klitigc.
208
Per cent of max response
Time 10 peak 01 response 51
