European Jownof of P~a~~ac~o~, 196 (1991) 77-83 0 1991 Elscvier Science Publishers B.V. OOM-2999/91/$03.50 ADONIS 0014299991002830
77
EIP 51806
Francis
1. Achike and Soter Dai
Departmentof Pharmacology, FacUrry of Medicine, Vniversiry of Hong Kong 5 SassoonRoad Hong Kong
Received9 August 1990. revised MS received 19 November 1990, accepted 22 January 1991
Langendorff preparations of Sprague-Dawley rat hearts were perfu,xd with calcium-free ICrebs solution of pH 7.48 (contro& 7.26 (acidosis) or 7.69 (alkalosis) containing either adrenaline or potassium. The responses of the force of contraction, coronary perfusion pressure and heart rate to graded doses of calcium preceded by a single dose of verapamil were measured. Contractile responsiveness to caIcium was reduced during acidosis in both adrenaline- and ~tassium-stimulate hearts but was increased or reduced during alkalosis with adrenaline- or potassium stimulation, respectively. The efficacy of verapami! as a calcium antagonist increased during acidosis or alkalosis in both adrenaline- and potassium-stimdated hearts. In conclusion, acidosis or alkalosis inhibits atrium-stimulate ~ntractions of the heart and enhances the effects of verapamil on potassium- and adrenaline-mediated contractions. Acidosis inhibits and alkalosis enhances adrenaline-stimulated contractions. Acidosis; Alkalosis; Verapamit; Ca** channels; Heart (rat)
1. Introdudion Several authors have demonstrated the beneficial effects of calcium channel blockers on ischaemic organ damage in both animal (Kazda et al., 1982; Milde et al., 1986) and human (Beckstead et al., 1918) studies. Ischaemic conditions are commonly associated with blood gas/pH abnormalities. It has been shown that changes in blood gas/pH will alter ~diova~ul~ responses to certain drugs (Dai and Wong, 1985; MacLean and Hiley, 1988). Achike and Dai (1990a) have demonstrated altered cardiovascular responses to nifedipine or verapamil in hyperventilated and hypoventilated rats. Subsequent studies (Achike and Dai, 1989; 199Ob) revealed that pH changes altered the sensitivity of cardiovascular tissues to calcium channel blockers, a finding that is in agreement with those of other workers (Smith and Briscoe, 1985). Trans-plasmalemmal calcium inflow, which triggers the in~a~llular process of contraption, is an important step in eliciting contraction of cardiac and smooth muscle. It is generally agreed that this process can be regulated by a change in membrane potential or by receptor activation (Bolton, 1979, Van Breemen et al.,
Correspondence to: FL Achike, Department of Pharmacology, Faculty of Medicine, University of Hong Kong, 5 &soon Road, Hong
Kong.
1979). Bolton (1979) classified the calcium channels as potentialand receptor-operated calcium channels (POCs and ROCs, respectively). It has been demonstrated that the binding of certain calcium channel blockers shows similar characteristics in membrane preparations from cardiac and smooth muscle tissues (Janis et al., 1982; Sarmiento et al., 1984) and that a correlation exists between binding and negative inotropic potency (Janis et al., 1984). The influx of calcium stimulated by noradrenaline or potassium in the rabbit aorta has been shown by Meisheri et al. (1981) and Meisheri and Van Breemen (1982) to be selectiveiy inhibited by organic calcium antagonists. Therefore, the possibility exists that a similar phenomenon occurs in cardiac tissue. The purpose of this study, therefore, was to observe the relative contributions of putative ROCs and FQCs to the earlier reported changes in sensitivity to verapamil (Achike and Dai, F99Ob) during acidosis and alkalosis.
2. Materials and metbds 2. I. Isolated heart preparations Male Sprague-Dawley rats, weighing 300-350 8, were killed by a sharp blow on their heads. Their hearts were quickly removed, mounted on a Langendorff perfusion apparatus, and immediately perfused through the aorta
sohttion of the following comNaCI 117.9; KC1 4.7; NaH?PO, 1.2: NaHCO, 25.0; glucose 1r.1 and 1.3. The pH of the solution was 7.48: it with a gas misture of 35% 0,. 5% CO, hout the esperiment and kept at a temperature of . Perfusion was by means of a peristaltic pump Minipuls 2. Gilson) at a fixed rate of 12 ml/min. A with a silk line was applied to the apex of the and then connected to an isometric myograph (Nsrco Bio-systems; Model F-60) for the measurement of force of contraction (FC). Heart rate (HR) was dL&uced from the frequency of the contraction. The sensitivity of the myograph was fixed throughout the experiments to enable adequate comparison within and between groups. A Stothsm P231D pressure transducer was connected to the side arm of the perfusing cannula for measuring the coronary perfusion pressure (PP). Ail the measured parameters were displayed on a physiograph (Narco Bio-systems). Krebs
_._. ’ ’ Effects of acidosis und ulkulosis on adrenaline-
or
potussiul,t-i~tdtI~.ed contraction
After a 25 to SO-min equilibration period. the preparations were subjected to continuous perfusion with calcium-free Krebs solution to whcih was added 4 x IO-' M EDTA: the composition of the solution was further as described above. The calcium-free Krebs solution contained either adrenaline (Sigma) (0.5 pm) or additional 5.0 mM KCI. A preliminary experiment had shown that the chosen concentrations of adrenaline or potassium induce contractions (go-100%) greater than those obtained by perfusion with normal Krebs. Higher do,xs of potassium tended to stop the contraction. The perfusion !huid -was maintained at either the control pH (7.48 + 0.02) or was made acidic (pH 7.26 + 0.01) or alkaline (pH 7.69 k 0.01). Acidosis was induced by making the perfusion medium acidic by the addition of 0.3 ml of 1 M HCI to every 200 ml of Krebs solution every 3-4 min. Preliminary tests showed that the pH of the gassed Krebs solution remained acidic for only 3-4 min and soon returned to normal after a single addition of HCI. Therefore. frequent addiuon of HCI to the perfusate was necessary to maintain the isolated hearts in a state of acidosis. Alkalosis was induced by making the per&ate alkaline by the addition of 4 ml of 1 M NaHCO, to every htre of the calcium-free Krebs solution in the reservoir. The hearts soon stopped contracting on changing to calcium-free perfusion. Increasing bolus doses of CaCI, (0004x. 0.008x. 0.015x. 0.03x. 0.06x. 0.12x. 0.24X, 0.48 X. 0.96 x, 1.91 x and 3.83 x 1O-5 M), which were preceded by an equivalent volume (0.1 ml) of calcium-free Krebs solution, were then administen:d to the hearts through a fine internal polythene tube
which terminated at the tip of the aortic cannula. The CaCl 2 was injected within 5 s after the previous contraction. This gave a dose interval of 20-35 s. with a shorter duration of contraction with the lower doses. 2.3. Effects of acidosis and alkalosis adrenaline-
or
on the sensitivities of
potassiunt-stimulated
contractions
to
verapunti1
The effect of acidosis or alkalosis on the sensitivity of adrenaline- or potassium-stimulated contractions to verapnmil was examined in separate preparations in which dose-response curves to calcium were made after the administration of 0.3 pg of verapamil (Knoll AG, Ludwigshafen) to each heart under control, acidic or alkaline conditions. The dose-response curves made during acidosis or alkalosis were compared with those obtained for the control group. 2.4. Preparation
of drugs
Verapamil and adrenaline were each dissolved in NaCI v/w (saline). They were freshly prepared before use and care was taken to prevent photodegradation. Ascorbic acid (10m4 M) was added to the adrenaline-containing calcium-free Krebs solution.
0.9%
2.5. Statistical analysis Maximal changes in FC, PP and HR following every dose of calcium were recorded. An FC dose-response curve was obtained for each preparation. The dose of calcium needed to produce 50% of the maximum contraction (EC,,) and the maximum force of contraction (FC,,, ) were obtained for each heart. The mean values of the EC,, and FC,,, for each experimental group were determined. Data obtained from the acidosis or alkalosis groups were compared with those obtained from the control group on a point-to-point basis by using Student’s unpaired t-test. The significance of differences was fixed at a minimum level of P < 0.05.
3. Results 3.1. Effect of perfusion with calcium-free
Krebs solution
The initial FC, PP and HR values before exposure of the hearts to calcium-free conditions were 7.25 + 0.18 g, 14.93 + 0.52 mm Hg and 335.00 f 5.98 beats/mm, respectively (n = 84). There were no significant differences in these values among the various experimental groups. On perfusion with calcium-free Krebs solution, cardiac contractions soon stopped and, consequently, the FC and HR values became zero. However, the PP value remained essentially unchanged. The time taken
79
from the onset of calcium-free perfusion to the stoppage of cardiac contractions in adrenalin~stimulat~ hearts was 11.89 f 0.24, 9.03 f 0.17 and 12.11 f 0.20 min for the control, acidosis and alkalosis groups, respectively; the corresponding figures for the potassium-stimulate hearts were 9.08 f 0.09.8.51 f 0.07 and 8.80 + 0,06 min, respectively (n = 7 for each group). Compared with their respective controls, there were significant decreases in the stopage time during acidosis (P c 0.001) in adrenaline-stimulated hearts and during acidosis (P c 0.001) or alkalosis (F c 0.01) in potassium-stimulated hearts. 3.2. Eflects of acidosis and alkalosis on adrenaline- and potassium-stimulated contractions Figure 1 shows the FC, PP and HR responses of adrenaline-stimulated hearts to graded doses of calcium under control, acidic and alkaline conditions. As indicated in table 1, the EC,, values derived from the FC dose-response curves (fig. 1) were 3.44 x , 7.90 x and 1.77 x lo-’ M for the control, acidosis and alkalosis groups, respectively. Compared with the control group, the acidosis group was 2.30 times and the alkalosis group 0.51 times less sensitive to the contractile effect of calcium (table 1). In the dose range of calcium used, acidosis and alkalosis did not seem to have an effect on adrenaline-stimulated PP responses (fig. 1). The HR responses during acidosis (fig. 1) tended to confirm the in~bito~ effect of acidosis on adrenalin~stimuiated cardiac responses to calcium as observed for the FC responses. Significant decreases in the HR response to calcium were noted at 0.008 x and 0.03 x 10-s M of calcium. The effect of alkalosis was not distinguishable from that of the control. FC, PP and HR responses to calcium in potassiumstimulated hearts are shown in fig. 2. The EC,, values of the FC dose-response curve were 14.75 x , 44.97 X and 19.16 x lo-’ M of calcium for the control. acidosis and
aikalosis groups, respectively. This makes the acidosis group 3.05 times and the alkalosis group 1.30 times kss sensitive to calcium than the normal potassium-stimulated hearts. The results shown in fig. 2 suggest that acidosis tends to attenuate the increase in PP seen with increasing doses of calcium in the controls. Significant differences (P -z 0.05) were recorded at 0.96 x and 0.48 X lo-’ M calcium. Alkalosis, however. appears to have had an effect on PP quite similar to that observed in the controls. HR responses in the ~tassium-stimulate hearts showed a pattern of dose-related increases in all the experimental conditions (fig. 2). However, for the alkalosis and more so for the acidosis group there was a tendency towards attenuation of the HR responses. This was significant (P ( 0.01) at O-03 x 10m5 M calcium for the alkalosis group and for all the doses except the 0.12 X 10e5 M calcium dose in the acidosis group. 3.3. Effect of verapami~ on adrenaline-stimulated contraction Figure 3 shows the FC, PP and HR responses to calcium in adrenaline-stimulated, verapamil-treated hearts. FC showed a dose-dependent increase in ail the experimental conditions, with EC,, vaiues of 3.63 X , 21.20 x and 3.84 x lo-’ M calcium for the control. acidosis and alkalosis groups, respectively. Verapamil was 5.84 times more effective in blocking the adrenaline-mediated FC during acidosis than under control conditions. Atkalosis did not seem to alter the efficacy of verapamil, with a sensitivity index of 1.06. The PP response to verapamil in the normal group was a dosedependent increase but verapamil was less effective in attenuating the PP increases during acidosis: there were significant differences from the control hearts at doses of 0.008 x and 6.52 x lo-’ M calcium (fig. 3). In the alkalosis group there was also a tendency to greater increases in PP than in the controls, particularly at the higher doses of calcium; however, these increases were
TABLE 1
.. .
.
EC,,, sensltlvrty Index and FCW, values for adrenaline- and Potassium-stimulated, verapamil- or vehicle-treated rat hearts. EC, and FC-: defined in the text; the values are the means& SEM., n = 7 for each group. Sensitivity index: determined by dividing the EC, values of the experimental groups with those of iheir respective controts. Potassium-stimulated contraclions
Adrenaline-stimulated contractions EC,, (x10-‘)M ~er~~rnif-r~~r~~ Normat 3.63 f 0.87 Acidosis 21.2Ok6.21 Alkalosis 3.84f 1.25
EC,, (x10-‘)M
Sensitivity index
1.06
14.55 f 1.77 17.47 f 1.04 12.84 f 2.71
16.26f2.15 66.95 f 9.08 35.s2*a.30
4.112 2.20
la.96 + 1.73 15.40+0.65 18.75 f 2.42
2.30 0.51
12.11 f 3.73 15.84f2.14 12.74 f 3.34
14.75 f 2.93 44.97 f 7.62 19.16i 3.89
3.05 1.30
18.01 io.49 15.20f2.17 20.59 f 2.04
Sensitivity index
PC,,
5.84
(g)
FC,,
(g) .-
Vehicle-lreared
Normal Acidosis Alkalosis
3.44+ 1.63 7.90 f 2.79 1.77f0.59
alkalosis groups of potassium-stimulated hearts, respectively (table 1). Thus verapamil was 4.12 times more effective in blocking potassium-stimulated FC responses in the acidosis group than iu the control group and 2.2 times more effective in the alkalosis group. The PP response in the presence of verapamil was quite similar in all the groups for the calcium dose range tested; there was a significant increase in PP in the acidosis group at a calcium dose of 0.03 x 10 -.’ M (fig. 4). The HR
t 1.1
6.1
6.1 -4
5.6
I
I
6.0
6.6
lC6l
Fig. I. Increases in the ferce of contraction (FC) (upper panel). coronary perfusion pressure (PP) (middle panel) and heart rate (HR) in response to graded doses of calcium under normal acidic (A) and alkaline (0) conditions in vehicle-treated, mulated rat hearts. n = 7 for each group. The values plotted are the means* S.E.M. * P -z 0.05 when compared with the corresponding control values.
not statistically significant. The HR response in each of the experimental conditions was a dose-dependent increase. The bradycardic effect of verapamil was more pronounced in the acidosis group than in the control group, whereas its effect in the alkalosis group was similar to that observed in the control group (fig. 3). 3.4. E~!eci of verapamil on potassium-stimulated contraction EC, values of 16.26 x ,66.95 x and 35.82 x lO-’ M calcium were obtainti for the control, acidosis and
, 6.6
a
6.1
5.6
I
5.0
6.6
-lag IC6l
Fig. 2. Increases in the force of contraction (FC) (upper panel), coronary perfusion pressure (PP) (middle panel) and heart rate (HR) (IOWY panel) in response to graded doses of calcium under normal ), acidic (A) and alkaline (0) conditions in vehicle-treated, potassium-stimulated rat hearts. n = 7 for each group. The values plotted are the means& S.E.M. * P -z 0.05. ** P < 0.01. * * * P < 0.001 when compared with the corresponding control values.
response in all the experimental conditions was dependent increase. However, verapamil had a bradycardic effect in the acidosis and alkalosis (more significantly so in acidosis) than in the group (fig. 4).
a dosegreater groups control
4. Discussion Cardiac tissue sensitivity to calcium was reduced during acidosis for both adreata!ine- and potassium-
Fig. 4. increases in the force of contraction (FC) (upper panel). coronary perfusion pressure (PP) (middle panel) and heart rate (HR) (lower panel) in response to graded doses of cafcium under normal ). acidic (A) and alkaline (0) conditions in verapamiftreated. potassium-stimulated rat hearts. n = 7 for each group. The values plotted are me means 1: S.E.M. * P -z 0.05; * * P < 0.01. * * * P c 0.001 when compared with the corresponding comrol values.
Fig. 3. Increases in the force of contraction (FC) (upper panel). coronary perfusion pressure (PP) (middle panel) and heart rate (HR) (lower panel) in response to graded doses of calcium under normal ), acidic {A) and alkaline (0) conditions in verapamiltreated, ad~na~ine-s~irnu~at~ rat hearts. n = 7 for each group. The values plot&d are the means f S.E.M. * P < 0.05. * * P c: 0.02. * * * P < 0.01 when compared with the corresponding control values.
stimulated contractions (figs. 1 and 2). with EC,, values 2.3 and 3.05 times their respective controls (table 1). This indicates that one or more of the various steps leading to contraction have been down-regulated. The exact mechanism involved cannot be identified from the present results. However, several authors have attributed the reduction by acidosis of cardiac or smooth muscle FC to a reduction in trans-membrane calcium
er et al.. 1979; Ebeigbe. 1982). increased r calcium to sarcoplasmic reticuwartz. 1970). or the deIc proteins to calcium 1978; Allen and Orchard. 1983). fated contractions involve mainly the zation of calcium (Peiper et al.. 1971; wan. 1982: Thorin-Trescases et al.. n the reduction in adrenaline-induced contracng acidosis will more likely be an intracellular tbis is so, then it could be due to a decrease in cytosshc free calcium resulting from the increased binding of intrace~lul~ calcium to SR or/and the desensitization of the contractile proteins to calcium. Potassiuced contractions involve the mobilization of War calcium (Peiper et al., 1971: Fleckenstein, 1977; Cauvin et al.. 1953). which in turn may induce a release of intmcellular calcium (Fabiato and . 1979). The reduced potassium-induced FC during acidosis may therefore be the result of a disturbance of extracellular calcium mobilization or of the intrace%tlsr mechanisms which could be desensitized by an increased calcium inflow during acidosis in cardiac mu_ccle(Allen and Orchard. 1983). The identification of hanisms involved will require further studies. an EC, vahre 0.51 times that of the control group, adrenaline-induced contractions were more sensitive to calcium during alkalosis. This is consistent with the findings of Peiper et al. (1971) who showed that noradrenaline- but not potassium-induced contractions decreased with decreasing PH. 1t could be that alkalosis enhances the levels of cytosolic free calcium by decreasing tbe binding of calcium by the SR, as has been demonstrated in cardiac and skeletal muscles by Nakamaru and Schwartz (1970). The potassium-induced FC. however, tended to decrease during alkalosis (EC,, value 1.30 times that of control). If the intracellular components of events leading to contraction are enhanced during alkalosis, as has been suggested by several authors (Nakamaru and Schwartz, 1970; Allen and Orchard, 1983) then it could be that alkalosis inhibits some extracellular mechanisms by which potassium-induced contractions are effected. This suggestion is supported by the increased sensitivity to verapamii observed in potassium-stimulated hearts during alkalosis !fig. 3). It is remarkable that cardiac tissue sensitivity to calcium was reduced in the same groups in which significant reductions were recorded in the time between onset of perfusion with calcium-free Krebs solution and stoppage of cardiac contraction (section 3.1.). This may reflect the effectiveness of acidosis or alkalosis in enhancing the nlegative inotropic effect of a lack of extracellular calcium during potassium-induced contractions. The reduction or increase in contraction time during acidosis or alkalosis, respectivley, in the adrenaline-stimulated hearts may be further evidence of the
desensitization or sensitization of the intracellular contractile process by acidosis or alkalosis. respectively. Within the pH range studied, the dynamics of the coronary vasculature were not altered in adrenalinestimulated hearts but there was a tendency towards reduced sensitivity to calcium during acidosis in the potassium-stimulated hearts (fig. 1). HR responses during acidosis in both adrenaline- and potassium-stimulated hearts tended to decrease; a further manifestation of the depressant effect of acidosis. A similar tendency in the potassium-stimulated hearts during alkalosis further suggests an inhibitory effect of aikalosis on POC. The sensitivity of the FC to verapamil in adrenalineand potassium-stimulated hearts during acidosis was increased 5.84 and 4.12 times, respectively. This result agrees with that of Smith and Briscoe (1985) who showed that acidosis caused an S-fold sensitization of the cat papillary muscle to the negative inotropic effect of verapamil. The present results, however, have shown that the potentiating effect of verapamil during acidosis is applicable to adrenaline- and potassium-induced contractions. Comparison of the EC,, values for alkalotic, verapamil-treated, and vehicle-treated adrenaline-stimulated hearts gave a ratio of 2.17, which compares with a ratio of 1.06 for the normal (control) hearts and conEirnis the 2.05fold increase in sensitivity to veraparnil during alkalosis. A similar calculation (data not shown) for all the other experimental groups further supports our observation that acidosis or alkalosis sensitizes adrenaline- or potassium-induced cardiac contractions to verapamil. In the potasisum-stimulated hearts, sensitivity to verapamil increased 2.2-fold during alkalosis. A trend seems to emerge that suggests that either acidosis or alkalosis sensitizes adrenaline- or potassium-stimulated rat hearts to verapamil. The changes in the responses to verapamil may not be attributable to the ionization state of this drug, which has a pK, value of 8.75 (Hasegawa et al., 1984), given the range of pH changes in the experiments. The same pattern of effects would noi he expected in acidosis as in alkalosis if the pH-induced ionization state of the drug were responsible for the effects observed. Smith and Briscoe (1985) also suggested that there is no increased sarcolemmal binding of ver ,pamil during acidosis. It is possible that the FC deprerz.ant effect of acidosis may potentiate the effects of verapamil. Smith and Briscoe (1985) proposed that when a small amount of sarcolemmal calcium entry has a major effect on intracellular calcium release, the tissue should be sensitive to a calcium entry blocking drug. Applying this proposal to the present results, the increased sensitivity to verapamil in the adrenalinestimulated hearts during aikalosis could be attributable to the combined cytosolic calcium-enhancing effect of adrenaline (Allen and Blinks, 1978) and alkalosis (Nakamaru and Schwartz, 1970). Similarly, the contraction-enhancing effect of alkalosis may partly explain the
83
increased sensitivity of potassium-stimulated hearts to verapamil. The PP responses in both adrenaline- and potassium-stimulated hearts were essentially similar to those of their respective controls, which suggests that, within the dose range of verapamil tested. the levels of acidosis or alkalosis induced may not alter PP responses. The HR responses showed a pattern that could generally fit into the explanations for the FC responses. In conclusion, the present study, while confirming the results of previous work that acidosis inhibits the contractile process, has shown that acidosis inhibits both adrenaline- and potassium-induced cardiac contractions. The results afso suggest that alkalosis may inhibit putative cardiac POCs and, like acidosis, sensitizes putative ROCs or POCs in the rat heart to verapamil. The m~hanism~ involved in these observations are not clear: further studies are needed.
Acknowledgements The authors gratefully acknowledge the helpful comments of Professor C.W. O@e and the technical assistance of Miss S.Y.N. Lee.
References Achike, F.I. and S. Dai. 1989, An abnormal blood pH affects the cardiovascular responses to verapamil, in: 4th International Symposium on Calcium Antagonists: Pharmacology and Clinical Research, Florence (Italy) (Fondazione Giovanni Lorenxini) p. 233. Achike, F.I. and S. Dai. 199Oa, Cardiovascular responses to verapamii and nifedipine in hypoventilated and hyperventilated rats, Br. J. Pharmacol. 100,102. Achike. El. and S. Dai. 199Ob. Responses of the isolated rat heart tr calcium channel blockers are altered by pH changes, FASEB J. 4, A869. Allen. D-0. and J.R. Blinks, 1978. Calcium transients in aequorin-injetted frog cardiac muscle, Nature 273.509. Allen, D.G. ard C.H. Orchard, 1983, The effect of pH on intracellular calcium transients in mammalian heart muscle, J. Physiol. 355, 555. Beckstead, J.E., W.A. Tweed, J. Lez and W.L. Mackeen, 1978, Cerebral blood flow and metabolism in man following cardiac arrest, Stroke 9. 569. Bolton, T.B.. 1979, Mechanisms of action of transmitters and other substances on smooth muscle. Physiol. Rev. 59.606. Cauvin. C., R. Loutxenhiser and C. Van Breemen, 1983, Mechanism of Ca+ antagonist induced vasodilatation, Ann. Rev. Pharmacol. Toxicol. 23, 373. Dai. S. and Y.H. Wang. 1985, Effects of hypoventilation on the cardiovascular responses of rats to adrenaline and acetylcholine, Pharmacology 30.314. Ebeigbe, A.B., 1982. Influence of hypoxia on contractility and calcium uptake in rabbit aorta, ~pe~entia 38, 935.
Fabiato. A. and F. Fabiato. 1978. Effects of pH on the myofilamenb and the sarcoplasmic reti~lum of skinned cells from cardiac and skeletal muscles, J. Physiol. 276, 233. Fabiato. A. and F. Fabiato, 1979. Calcium and cardiac excitation-_ traction coupling, Ann. Rev. Physiol. 41,473. Fleckenstein, A.. 1977, Specific pharmacology of calcium in myocardium.cardiac pacemakers and vascular smooth muscle. Ann. Rev. Pharmacol. Toxicol. 17, 149. Hasegawa, J., T. Fuji% Y.. Hay&i. K.. fwamoto and J. Watana&, 1984. pKa determination of verapamil by liquid-liquid partition, J. Pharmacol. Sci. 73,442. Heaslip, R.J. and R.G. Rahwan, 1982, Evidence for the existence of two distinct pools of intracellular calcium in the rat aorta accessible to mobilization by norepinephrine, J. Pharmacol. Exp. Ther. 221.7. Janis. RA., SC. Maurer, J.C. Sarmiento, G-T. Bolger and DJ. Triggle. 1982, Binding of [‘Hlnimodipinc to cardiac and smooth muscle membranes, European J. Pharmacol. 82,191. h!h. R-A., J.G. Sarmiento. SC. Maurer, G.T. Bolger and DJ. Triggle, 1984. Characteristics of the binding of [ ‘Hlnitrendipine to rabbit ventricular membranes: modificaiton by other Ca2+ channel antagonists and by the Ca2’ channel agonist Bay K8644_ J. Pharamcot. Exp. Ther. 231.8. Kazda. S.. B. Garthoff, H.P. Krause and K. Schlobmann, 1982, Cerebrovascular effects of the calcium antagonistic dihydropyridine derivative n~~ipine in animal experiments, Arzneim. Forsch. 32. 331. MacLean. M.R. and C.R. Hiley, 1988, Effects of artificial respiratory volume on the cardiovascular responses of an a, and an aa-adrenoceptor agonist in the air-ventilated pithed rat, Br. J. Pharmacol. 93. 781. Meisberi, K.D.. 0. Hwang and C. Van Breemen. 1981. Evidence of two separate Ca2+ pathways in smooth muscle plasmalemma 1. Membr. Biol. 59. 19. Meisheri. K.D. and C. Van Breemen. 1982. Effects of /3-adrenergic stimulation on calcium movem~~ in rabbit aortic smooth muscle: relationship with cyclic AMP, J. Physiol. 331,429. Milde, L.N., J.H. Milde and J.D. Michenfelder. 1986. Delayed treatment with nimodipine improves cerebral blood flow after complete cerebral ischaemia in the dog. J. Cereb. Blood ROW Metab. 6, 332. Nakamaru, Y. and A. Schwartz, 1970, Possible control of intracellular calcium meta~lism by (H + 1: sarcoplasmic reticulum of skekti and cardiac muscle, Biochem. Biophys. Res. Commun. 41.4. 830. Nayler, W.G., P.A., Poole-Wilson and A. Williams. 1970, Hypoxia and calcium, J. Mol. Ceil Cardiol. 11,683. Peiper, U.. I_. Griebel and W. Wende, 1971, Activation of vascular smooth muscle of rat aorta by noradrenaline and depolarization: two different mechanisms, PBiigers Arch. 33% 74. Sarmiento, J.G.. R.A. Janis. A.M. Katz and DJ. Triggle. 1984, Corn parison of the higb affinity binding of calcium channel blocking drugs to vascular smooth muscle and cardiac sarcolemmal membranes, Biochem. Pharmacol. 33.3119. Smith. H.J. and M.G. Briscoe, 1985. The relative sensitization by acidosis of five calcium blockers in cat papillary muscles, J. Mol. Cell Cardiol. 17, 799. Tho&t-Tmscases, N., L. Oster. J. Atkinson and c. Capdevilie. 199% Norepinephrine and serotonin increase the VasoiXnstriCtor response of the perfused rat tail artery to changes in fytosOliC Ca2 +, European J. Pharmacol. 179,469. Van Breemen, C., P. Aaronson and R. Loutxenhiser, 1979. Sodiumcalcium interactions in mammalian smooth muscles.Phamxd. Rev. 30,167.
77
EIP 51806
Francis
1. Achike and Soter Dai
Departmentof Pharmacology, FacUrry of Medicine, Vniversiry of Hong Kong 5 SassoonRoad Hong Kong
Received9 August 1990. revised MS received 19 November 1990, accepted 22 January 1991
Langendorff preparations of Sprague-Dawley rat hearts were perfu,xd with calcium-free ICrebs solution of pH 7.48 (contro& 7.26 (acidosis) or 7.69 (alkalosis) containing either adrenaline or potassium. The responses of the force of contraction, coronary perfusion pressure and heart rate to graded doses of calcium preceded by a single dose of verapamil were measured. Contractile responsiveness to caIcium was reduced during acidosis in both adrenaline- and ~tassium-stimulate hearts but was increased or reduced during alkalosis with adrenaline- or potassium stimulation, respectively. The efficacy of verapami! as a calcium antagonist increased during acidosis or alkalosis in both adrenaline- and potassium-stimdated hearts. In conclusion, acidosis or alkalosis inhibits atrium-stimulate ~ntractions of the heart and enhances the effects of verapamil on potassium- and adrenaline-mediated contractions. Acidosis inhibits and alkalosis enhances adrenaline-stimulated contractions. Acidosis; Alkalosis; Verapamit; Ca** channels; Heart (rat)
1. Introdudion Several authors have demonstrated the beneficial effects of calcium channel blockers on ischaemic organ damage in both animal (Kazda et al., 1982; Milde et al., 1986) and human (Beckstead et al., 1918) studies. Ischaemic conditions are commonly associated with blood gas/pH abnormalities. It has been shown that changes in blood gas/pH will alter ~diova~ul~ responses to certain drugs (Dai and Wong, 1985; MacLean and Hiley, 1988). Achike and Dai (1990a) have demonstrated altered cardiovascular responses to nifedipine or verapamil in hyperventilated and hypoventilated rats. Subsequent studies (Achike and Dai, 1989; 199Ob) revealed that pH changes altered the sensitivity of cardiovascular tissues to calcium channel blockers, a finding that is in agreement with those of other workers (Smith and Briscoe, 1985). Trans-plasmalemmal calcium inflow, which triggers the in~a~llular process of contraption, is an important step in eliciting contraction of cardiac and smooth muscle. It is generally agreed that this process can be regulated by a change in membrane potential or by receptor activation (Bolton, 1979, Van Breemen et al.,
Correspondence to: FL Achike, Department of Pharmacology, Faculty of Medicine, University of Hong Kong, 5 &soon Road, Hong
Kong.
1979). Bolton (1979) classified the calcium channels as potentialand receptor-operated calcium channels (POCs and ROCs, respectively). It has been demonstrated that the binding of certain calcium channel blockers shows similar characteristics in membrane preparations from cardiac and smooth muscle tissues (Janis et al., 1982; Sarmiento et al., 1984) and that a correlation exists between binding and negative inotropic potency (Janis et al., 1984). The influx of calcium stimulated by noradrenaline or potassium in the rabbit aorta has been shown by Meisheri et al. (1981) and Meisheri and Van Breemen (1982) to be selectiveiy inhibited by organic calcium antagonists. Therefore, the possibility exists that a similar phenomenon occurs in cardiac tissue. The purpose of this study, therefore, was to observe the relative contributions of putative ROCs and FQCs to the earlier reported changes in sensitivity to verapamil (Achike and Dai, F99Ob) during acidosis and alkalosis.
2. Materials and metbds 2. I. Isolated heart preparations Male Sprague-Dawley rats, weighing 300-350 8, were killed by a sharp blow on their heads. Their hearts were quickly removed, mounted on a Langendorff perfusion apparatus, and immediately perfused through the aorta
sohttion of the following comNaCI 117.9; KC1 4.7; NaH?PO, 1.2: NaHCO, 25.0; glucose 1r.1 and 1.3. The pH of the solution was 7.48: it with a gas misture of 35% 0,. 5% CO, hout the esperiment and kept at a temperature of . Perfusion was by means of a peristaltic pump Minipuls 2. Gilson) at a fixed rate of 12 ml/min. A with a silk line was applied to the apex of the and then connected to an isometric myograph (Nsrco Bio-systems; Model F-60) for the measurement of force of contraction (FC). Heart rate (HR) was dL&uced from the frequency of the contraction. The sensitivity of the myograph was fixed throughout the experiments to enable adequate comparison within and between groups. A Stothsm P231D pressure transducer was connected to the side arm of the perfusing cannula for measuring the coronary perfusion pressure (PP). Ail the measured parameters were displayed on a physiograph (Narco Bio-systems). Krebs
_._. ’ ’ Effects of acidosis und ulkulosis on adrenaline-
or
potussiul,t-i~tdtI~.ed contraction
After a 25 to SO-min equilibration period. the preparations were subjected to continuous perfusion with calcium-free Krebs solution to whcih was added 4 x IO-' M EDTA: the composition of the solution was further as described above. The calcium-free Krebs solution contained either adrenaline (Sigma) (0.5 pm) or additional 5.0 mM KCI. A preliminary experiment had shown that the chosen concentrations of adrenaline or potassium induce contractions (go-100%) greater than those obtained by perfusion with normal Krebs. Higher do,xs of potassium tended to stop the contraction. The perfusion !huid -was maintained at either the control pH (7.48 + 0.02) or was made acidic (pH 7.26 + 0.01) or alkaline (pH 7.69 k 0.01). Acidosis was induced by making the perfusion medium acidic by the addition of 0.3 ml of 1 M HCI to every 200 ml of Krebs solution every 3-4 min. Preliminary tests showed that the pH of the gassed Krebs solution remained acidic for only 3-4 min and soon returned to normal after a single addition of HCI. Therefore. frequent addiuon of HCI to the perfusate was necessary to maintain the isolated hearts in a state of acidosis. Alkalosis was induced by making the per&ate alkaline by the addition of 4 ml of 1 M NaHCO, to every htre of the calcium-free Krebs solution in the reservoir. The hearts soon stopped contracting on changing to calcium-free perfusion. Increasing bolus doses of CaCI, (0004x. 0.008x. 0.015x. 0.03x. 0.06x. 0.12x. 0.24X, 0.48 X. 0.96 x, 1.91 x and 3.83 x 1O-5 M), which were preceded by an equivalent volume (0.1 ml) of calcium-free Krebs solution, were then administen:d to the hearts through a fine internal polythene tube
which terminated at the tip of the aortic cannula. The CaCl 2 was injected within 5 s after the previous contraction. This gave a dose interval of 20-35 s. with a shorter duration of contraction with the lower doses. 2.3. Effects of acidosis and alkalosis adrenaline-
or
on the sensitivities of
potassiunt-stimulated
contractions
to
verapunti1
The effect of acidosis or alkalosis on the sensitivity of adrenaline- or potassium-stimulated contractions to verapnmil was examined in separate preparations in which dose-response curves to calcium were made after the administration of 0.3 pg of verapamil (Knoll AG, Ludwigshafen) to each heart under control, acidic or alkaline conditions. The dose-response curves made during acidosis or alkalosis were compared with those obtained for the control group. 2.4. Preparation
of drugs
Verapamil and adrenaline were each dissolved in NaCI v/w (saline). They were freshly prepared before use and care was taken to prevent photodegradation. Ascorbic acid (10m4 M) was added to the adrenaline-containing calcium-free Krebs solution.
0.9%
2.5. Statistical analysis Maximal changes in FC, PP and HR following every dose of calcium were recorded. An FC dose-response curve was obtained for each preparation. The dose of calcium needed to produce 50% of the maximum contraction (EC,,) and the maximum force of contraction (FC,,, ) were obtained for each heart. The mean values of the EC,, and FC,,, for each experimental group were determined. Data obtained from the acidosis or alkalosis groups were compared with those obtained from the control group on a point-to-point basis by using Student’s unpaired t-test. The significance of differences was fixed at a minimum level of P < 0.05.
3. Results 3.1. Effect of perfusion with calcium-free
Krebs solution
The initial FC, PP and HR values before exposure of the hearts to calcium-free conditions were 7.25 + 0.18 g, 14.93 + 0.52 mm Hg and 335.00 f 5.98 beats/mm, respectively (n = 84). There were no significant differences in these values among the various experimental groups. On perfusion with calcium-free Krebs solution, cardiac contractions soon stopped and, consequently, the FC and HR values became zero. However, the PP value remained essentially unchanged. The time taken
79
from the onset of calcium-free perfusion to the stoppage of cardiac contractions in adrenalin~stimulat~ hearts was 11.89 f 0.24, 9.03 f 0.17 and 12.11 f 0.20 min for the control, acidosis and alkalosis groups, respectively; the corresponding figures for the potassium-stimulate hearts were 9.08 f 0.09.8.51 f 0.07 and 8.80 + 0,06 min, respectively (n = 7 for each group). Compared with their respective controls, there were significant decreases in the stopage time during acidosis (P c 0.001) in adrenaline-stimulated hearts and during acidosis (P c 0.001) or alkalosis (F c 0.01) in potassium-stimulated hearts. 3.2. Eflects of acidosis and alkalosis on adrenaline- and potassium-stimulated contractions Figure 1 shows the FC, PP and HR responses of adrenaline-stimulated hearts to graded doses of calcium under control, acidic and alkaline conditions. As indicated in table 1, the EC,, values derived from the FC dose-response curves (fig. 1) were 3.44 x , 7.90 x and 1.77 x lo-’ M for the control, acidosis and alkalosis groups, respectively. Compared with the control group, the acidosis group was 2.30 times and the alkalosis group 0.51 times less sensitive to the contractile effect of calcium (table 1). In the dose range of calcium used, acidosis and alkalosis did not seem to have an effect on adrenaline-stimulated PP responses (fig. 1). The HR responses during acidosis (fig. 1) tended to confirm the in~bito~ effect of acidosis on adrenalin~stimuiated cardiac responses to calcium as observed for the FC responses. Significant decreases in the HR response to calcium were noted at 0.008 x and 0.03 x 10-s M of calcium. The effect of alkalosis was not distinguishable from that of the control. FC, PP and HR responses to calcium in potassiumstimulated hearts are shown in fig. 2. The EC,, values of the FC dose-response curve were 14.75 x , 44.97 X and 19.16 x lo-’ M of calcium for the control. acidosis and
aikalosis groups, respectively. This makes the acidosis group 3.05 times and the alkalosis group 1.30 times kss sensitive to calcium than the normal potassium-stimulated hearts. The results shown in fig. 2 suggest that acidosis tends to attenuate the increase in PP seen with increasing doses of calcium in the controls. Significant differences (P -z 0.05) were recorded at 0.96 x and 0.48 X lo-’ M calcium. Alkalosis, however. appears to have had an effect on PP quite similar to that observed in the controls. HR responses in the ~tassium-stimulate hearts showed a pattern of dose-related increases in all the experimental conditions (fig. 2). However, for the alkalosis and more so for the acidosis group there was a tendency towards attenuation of the HR responses. This was significant (P ( 0.01) at O-03 x 10m5 M calcium for the alkalosis group and for all the doses except the 0.12 X 10e5 M calcium dose in the acidosis group. 3.3. Effect of verapami~ on adrenaline-stimulated contraction Figure 3 shows the FC, PP and HR responses to calcium in adrenaline-stimulated, verapamil-treated hearts. FC showed a dose-dependent increase in ail the experimental conditions, with EC,, vaiues of 3.63 X , 21.20 x and 3.84 x lo-’ M calcium for the control. acidosis and alkalosis groups, respectively. Verapamil was 5.84 times more effective in blocking the adrenaline-mediated FC during acidosis than under control conditions. Atkalosis did not seem to alter the efficacy of verapamil, with a sensitivity index of 1.06. The PP response to verapamil in the normal group was a dosedependent increase but verapamil was less effective in attenuating the PP increases during acidosis: there were significant differences from the control hearts at doses of 0.008 x and 6.52 x lo-’ M calcium (fig. 3). In the alkalosis group there was also a tendency to greater increases in PP than in the controls, particularly at the higher doses of calcium; however, these increases were
TABLE 1
.. .
.
EC,,, sensltlvrty Index and FCW, values for adrenaline- and Potassium-stimulated, verapamil- or vehicle-treated rat hearts. EC, and FC-: defined in the text; the values are the means& SEM., n = 7 for each group. Sensitivity index: determined by dividing the EC, values of the experimental groups with those of iheir respective controts. Potassium-stimulated contraclions
Adrenaline-stimulated contractions EC,, (x10-‘)M ~er~~rnif-r~~r~~ Normat 3.63 f 0.87 Acidosis 21.2Ok6.21 Alkalosis 3.84f 1.25
EC,, (x10-‘)M
Sensitivity index
1.06
14.55 f 1.77 17.47 f 1.04 12.84 f 2.71
16.26f2.15 66.95 f 9.08 35.s2*a.30
4.112 2.20
la.96 + 1.73 15.40+0.65 18.75 f 2.42
2.30 0.51
12.11 f 3.73 15.84f2.14 12.74 f 3.34
14.75 f 2.93 44.97 f 7.62 19.16i 3.89
3.05 1.30
18.01 io.49 15.20f2.17 20.59 f 2.04
Sensitivity index
PC,,
5.84
(g)
FC,,
(g) .-
Vehicle-lreared
Normal Acidosis Alkalosis
3.44+ 1.63 7.90 f 2.79 1.77f0.59
alkalosis groups of potassium-stimulated hearts, respectively (table 1). Thus verapamil was 4.12 times more effective in blocking potassium-stimulated FC responses in the acidosis group than iu the control group and 2.2 times more effective in the alkalosis group. The PP response in the presence of verapamil was quite similar in all the groups for the calcium dose range tested; there was a significant increase in PP in the acidosis group at a calcium dose of 0.03 x 10 -.’ M (fig. 4). The HR
t 1.1
6.1
6.1 -4
5.6
I
I
6.0
6.6
lC6l
Fig. I. Increases in the ferce of contraction (FC) (upper panel). coronary perfusion pressure (PP) (middle panel) and heart rate (HR) in response to graded doses of calcium under normal acidic (A) and alkaline (0) conditions in vehicle-treated, mulated rat hearts. n = 7 for each group. The values plotted are the means* S.E.M. * P -z 0.05 when compared with the corresponding control values.
not statistically significant. The HR response in each of the experimental conditions was a dose-dependent increase. The bradycardic effect of verapamil was more pronounced in the acidosis group than in the control group, whereas its effect in the alkalosis group was similar to that observed in the control group (fig. 3). 3.4. E~!eci of verapamil on potassium-stimulated contraction EC, values of 16.26 x ,66.95 x and 35.82 x lO-’ M calcium were obtainti for the control, acidosis and
, 6.6
a
6.1
5.6
I
5.0
6.6
-lag IC6l
Fig. 2. Increases in the force of contraction (FC) (upper panel), coronary perfusion pressure (PP) (middle panel) and heart rate (HR) (IOWY panel) in response to graded doses of calcium under normal ), acidic (A) and alkaline (0) conditions in vehicle-treated, potassium-stimulated rat hearts. n = 7 for each group. The values plotted are the means& S.E.M. * P -z 0.05. ** P < 0.01. * * * P < 0.001 when compared with the corresponding control values.
response in all the experimental conditions was dependent increase. However, verapamil had a bradycardic effect in the acidosis and alkalosis (more significantly so in acidosis) than in the group (fig. 4).
a dosegreater groups control
4. Discussion Cardiac tissue sensitivity to calcium was reduced during acidosis for both adreata!ine- and potassium-
Fig. 4. increases in the force of contraction (FC) (upper panel). coronary perfusion pressure (PP) (middle panel) and heart rate (HR) (lower panel) in response to graded doses of cafcium under normal ). acidic (A) and alkaline (0) conditions in verapamiftreated. potassium-stimulated rat hearts. n = 7 for each group. The values plotted are me means 1: S.E.M. * P -z 0.05; * * P < 0.01. * * * P c 0.001 when compared with the corresponding comrol values.
Fig. 3. Increases in the force of contraction (FC) (upper panel). coronary perfusion pressure (PP) (middle panel) and heart rate (HR) (lower panel) in response to graded doses of calcium under normal ), acidic {A) and alkaline (0) conditions in verapamiltreated, ad~na~ine-s~irnu~at~ rat hearts. n = 7 for each group. The values plot&d are the means f S.E.M. * P < 0.05. * * P c: 0.02. * * * P < 0.01 when compared with the corresponding control values.
stimulated contractions (figs. 1 and 2). with EC,, values 2.3 and 3.05 times their respective controls (table 1). This indicates that one or more of the various steps leading to contraction have been down-regulated. The exact mechanism involved cannot be identified from the present results. However, several authors have attributed the reduction by acidosis of cardiac or smooth muscle FC to a reduction in trans-membrane calcium
er et al.. 1979; Ebeigbe. 1982). increased r calcium to sarcoplasmic reticuwartz. 1970). or the deIc proteins to calcium 1978; Allen and Orchard. 1983). fated contractions involve mainly the zation of calcium (Peiper et al.. 1971; wan. 1982: Thorin-Trescases et al.. n the reduction in adrenaline-induced contracng acidosis will more likely be an intracellular tbis is so, then it could be due to a decrease in cytosshc free calcium resulting from the increased binding of intrace~lul~ calcium to SR or/and the desensitization of the contractile proteins to calcium. Potassiuced contractions involve the mobilization of War calcium (Peiper et al., 1971: Fleckenstein, 1977; Cauvin et al.. 1953). which in turn may induce a release of intmcellular calcium (Fabiato and . 1979). The reduced potassium-induced FC during acidosis may therefore be the result of a disturbance of extracellular calcium mobilization or of the intrace%tlsr mechanisms which could be desensitized by an increased calcium inflow during acidosis in cardiac mu_ccle(Allen and Orchard. 1983). The identification of hanisms involved will require further studies. an EC, vahre 0.51 times that of the control group, adrenaline-induced contractions were more sensitive to calcium during alkalosis. This is consistent with the findings of Peiper et al. (1971) who showed that noradrenaline- but not potassium-induced contractions decreased with decreasing PH. 1t could be that alkalosis enhances the levels of cytosolic free calcium by decreasing tbe binding of calcium by the SR, as has been demonstrated in cardiac and skeletal muscles by Nakamaru and Schwartz (1970). The potassium-induced FC. however, tended to decrease during alkalosis (EC,, value 1.30 times that of control). If the intracellular components of events leading to contraction are enhanced during alkalosis, as has been suggested by several authors (Nakamaru and Schwartz, 1970; Allen and Orchard, 1983) then it could be that alkalosis inhibits some extracellular mechanisms by which potassium-induced contractions are effected. This suggestion is supported by the increased sensitivity to verapamii observed in potassium-stimulated hearts during alkalosis !fig. 3). It is remarkable that cardiac tissue sensitivity to calcium was reduced in the same groups in which significant reductions were recorded in the time between onset of perfusion with calcium-free Krebs solution and stoppage of cardiac contraction (section 3.1.). This may reflect the effectiveness of acidosis or alkalosis in enhancing the nlegative inotropic effect of a lack of extracellular calcium during potassium-induced contractions. The reduction or increase in contraction time during acidosis or alkalosis, respectivley, in the adrenaline-stimulated hearts may be further evidence of the
desensitization or sensitization of the intracellular contractile process by acidosis or alkalosis. respectively. Within the pH range studied, the dynamics of the coronary vasculature were not altered in adrenalinestimulated hearts but there was a tendency towards reduced sensitivity to calcium during acidosis in the potassium-stimulated hearts (fig. 1). HR responses during acidosis in both adrenaline- and potassium-stimulated hearts tended to decrease; a further manifestation of the depressant effect of acidosis. A similar tendency in the potassium-stimulated hearts during alkalosis further suggests an inhibitory effect of aikalosis on POC. The sensitivity of the FC to verapamil in adrenalineand potassium-stimulated hearts during acidosis was increased 5.84 and 4.12 times, respectively. This result agrees with that of Smith and Briscoe (1985) who showed that acidosis caused an S-fold sensitization of the cat papillary muscle to the negative inotropic effect of verapamil. The present results, however, have shown that the potentiating effect of verapamil during acidosis is applicable to adrenaline- and potassium-induced contractions. Comparison of the EC,, values for alkalotic, verapamil-treated, and vehicle-treated adrenaline-stimulated hearts gave a ratio of 2.17, which compares with a ratio of 1.06 for the normal (control) hearts and conEirnis the 2.05fold increase in sensitivity to veraparnil during alkalosis. A similar calculation (data not shown) for all the other experimental groups further supports our observation that acidosis or alkalosis sensitizes adrenaline- or potassium-induced cardiac contractions to verapamil. In the potasisum-stimulated hearts, sensitivity to verapamil increased 2.2-fold during alkalosis. A trend seems to emerge that suggests that either acidosis or alkalosis sensitizes adrenaline- or potassium-stimulated rat hearts to verapamil. The changes in the responses to verapamil may not be attributable to the ionization state of this drug, which has a pK, value of 8.75 (Hasegawa et al., 1984), given the range of pH changes in the experiments. The same pattern of effects would noi he expected in acidosis as in alkalosis if the pH-induced ionization state of the drug were responsible for the effects observed. Smith and Briscoe (1985) also suggested that there is no increased sarcolemmal binding of ver ,pamil during acidosis. It is possible that the FC deprerz.ant effect of acidosis may potentiate the effects of verapamil. Smith and Briscoe (1985) proposed that when a small amount of sarcolemmal calcium entry has a major effect on intracellular calcium release, the tissue should be sensitive to a calcium entry blocking drug. Applying this proposal to the present results, the increased sensitivity to verapamil in the adrenalinestimulated hearts during aikalosis could be attributable to the combined cytosolic calcium-enhancing effect of adrenaline (Allen and Blinks, 1978) and alkalosis (Nakamaru and Schwartz, 1970). Similarly, the contraction-enhancing effect of alkalosis may partly explain the
83
increased sensitivity of potassium-stimulated hearts to verapamil. The PP responses in both adrenaline- and potassium-stimulated hearts were essentially similar to those of their respective controls, which suggests that, within the dose range of verapamil tested. the levels of acidosis or alkalosis induced may not alter PP responses. The HR responses showed a pattern that could generally fit into the explanations for the FC responses. In conclusion, the present study, while confirming the results of previous work that acidosis inhibits the contractile process, has shown that acidosis inhibits both adrenaline- and potassium-induced cardiac contractions. The results afso suggest that alkalosis may inhibit putative cardiac POCs and, like acidosis, sensitizes putative ROCs or POCs in the rat heart to verapamil. The m~hanism~ involved in these observations are not clear: further studies are needed.
Acknowledgements The authors gratefully acknowledge the helpful comments of Professor C.W. O@e and the technical assistance of Miss S.Y.N. Lee.
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Fabiato. A. and F. Fabiato. 1978. Effects of pH on the myofilamenb and the sarcoplasmic reti~lum of skinned cells from cardiac and skeletal muscles, J. Physiol. 276, 233. Fabiato. A. and F. Fabiato, 1979. Calcium and cardiac excitation-_ traction coupling, Ann. Rev. Physiol. 41,473. Fleckenstein, A.. 1977, Specific pharmacology of calcium in myocardium.cardiac pacemakers and vascular smooth muscle. Ann. Rev. Pharmacol. Toxicol. 17, 149. Hasegawa, J., T. Fuji% Y.. Hay&i. K.. fwamoto and J. Watana&, 1984. pKa determination of verapamil by liquid-liquid partition, J. Pharmacol. Sci. 73,442. Heaslip, R.J. and R.G. Rahwan, 1982, Evidence for the existence of two distinct pools of intracellular calcium in the rat aorta accessible to mobilization by norepinephrine, J. Pharmacol. Exp. Ther. 221.7. Janis. RA., SC. Maurer, J.C. Sarmiento, G-T. Bolger and DJ. Triggle. 1982, Binding of [‘Hlnimodipinc to cardiac and smooth muscle membranes, European J. Pharmacol. 82,191. h!h. R-A., J.G. Sarmiento. SC. Maurer, G.T. Bolger and DJ. Triggle, 1984. Characteristics of the binding of [ ‘Hlnitrendipine to rabbit ventricular membranes: modificaiton by other Ca2+ channel antagonists and by the Ca2’ channel agonist Bay K8644_ J. Pharamcot. Exp. Ther. 231.8. Kazda. S.. B. Garthoff, H.P. Krause and K. Schlobmann, 1982, Cerebrovascular effects of the calcium antagonistic dihydropyridine derivative n~~ipine in animal experiments, Arzneim. Forsch. 32. 331. MacLean. M.R. and C.R. Hiley, 1988, Effects of artificial respiratory volume on the cardiovascular responses of an a, and an aa-adrenoceptor agonist in the air-ventilated pithed rat, Br. J. Pharmacol. 93. 781. Meisberi, K.D.. 0. Hwang and C. Van Breemen. 1981. Evidence of two separate Ca2+ pathways in smooth muscle plasmalemma 1. Membr. Biol. 59. 19. Meisheri. K.D. and C. Van Breemen. 1982. Effects of /3-adrenergic stimulation on calcium movem~~ in rabbit aortic smooth muscle: relationship with cyclic AMP, J. Physiol. 331,429. Milde, L.N., J.H. Milde and J.D. Michenfelder. 1986. Delayed treatment with nimodipine improves cerebral blood flow after complete cerebral ischaemia in the dog. J. Cereb. Blood ROW Metab. 6, 332. Nakamaru, Y. and A. Schwartz, 1970, Possible control of intracellular calcium meta~lism by (H + 1: sarcoplasmic reticulum of skekti and cardiac muscle, Biochem. Biophys. Res. Commun. 41.4. 830. Nayler, W.G., P.A., Poole-Wilson and A. Williams. 1970, Hypoxia and calcium, J. Mol. Ceil Cardiol. 11,683. Peiper, U.. I_. Griebel and W. Wende, 1971, Activation of vascular smooth muscle of rat aorta by noradrenaline and depolarization: two different mechanisms, PBiigers Arch. 33% 74. Sarmiento, J.G.. R.A. Janis. A.M. Katz and DJ. Triggle. 1984, Corn parison of the higb affinity binding of calcium channel blocking drugs to vascular smooth muscle and cardiac sarcolemmal membranes, Biochem. Pharmacol. 33.3119. Smith. H.J. and M.G. Briscoe, 1985. The relative sensitization by acidosis of five calcium blockers in cat papillary muscles, J. Mol. Cell Cardiol. 17, 799. Tho&t-Tmscases, N., L. Oster. J. Atkinson and c. Capdevilie. 199% Norepinephrine and serotonin increase the VasoiXnstriCtor response of the perfused rat tail artery to changes in fytosOliC Ca2 +, European J. Pharmacol. 179,469. Van Breemen, C., P. Aaronson and R. Loutxenhiser, 1979. Sodiumcalcium interactions in mammalian smooth muscles.Phamxd. Rev. 30,167.
