Acta physiol. scand. 1976. 98. 266-268 From the Department of Physiology, University of Goteborg, Sweden
Quantitative Effects of External Calcium Concentration on Contraction of Rat Portal Vein Compared to Thoracic Aorta BY
MORLEY C. SUTTER'
The influence of external calcium concentration on the responses to noradrenaline (NA) has been examined in the thoracic aorta and portal vein of the rat. Cumulative concentration-response curves to NA have been produced at each of 6 concentrations of added calcium (ranging from 0 to 2.5 mM Ca2+)and the responses have been compared in two ways: 1) calculation of the ED,, for NA at each concentration of calcium; 2) the response to NA in the altered calcium has been expressed as a percentage of the response to the same concentration of N A in normal (2.5 mM) Ca2+.The results clearly show that alteration of external calcium concentration affects the EDSoand the magnitude of the responses to N A in both the aorta and the portal vein but that there are quantitative differences between the two tissues. The rat portal vein contracts and relaxes spontaneously, hence the effects of the several concentrations of external Ca2+on myogenic activity also could be examined in this tissue. One longitudinal portal vein preparation and one helical strip preparation of the thoracic aorta from each of 5 male Wistar rats (220-265 g) were mounted in organ baths for isometric recording at levels of passive force of 5 and 10 mN respectively. The force signals were electronically integrated. The calcium concentration of the fluid bathing the tissues was varied by adding CaCI, (0-2.5 mM) to a modified Krebs solution of the following composition in mM: NaCl 122; KCI 4.73; NaHCO, 15.5; KH,PO, 1.19; MgCI, 1.19; glucose 11,s; CaNa,-versenate 0.026. It was continuously bubbled with 4 per cent CO, in 0, and kept a t 38°C. The protocol of each experiment was t o allow the tissues to equilibrate in normal Krebs solution for at least 1 h and then to do a cumulative concentration-response curve to NA in concentrations from to M. The tissue was exposed to each concentration of NA for 2 min. After the highest dose of NA the fluid in the bath was changed to one having a different calcium concentration. The sequence was to proceed from fluid containing 2.5 m M added Ca2+ to one with n o added calcium, then to 0.1, 0.2, 0.4, 0.8 and back t o 2.5 m M Ca2+. Responses t o cumulative concentrations of N A were obtained at each level of calcium. Half a n hour was allowed for the tissues to equilibrate in each calcium concentration before doing each concentration-response curve. The response to each concentration of NA was calculated as the mean force developing during the two minute exposure period minus the mean force developed during the control period. Concentration-effect curves for NA a t each level of calcium were plotted as the least squares fit to a true hyperbolic function. The ED,, values in logarithmic form (log ED,,) were used for comparisons. On sabbatical leave from the Department of Pharmacology, University of British Columbia, Vancouver, Canada.
266
267
CALCIUM EFFECT ON PORTAL VEIN
PORTAL VFlN
'\\\
'P
Fig. 1. Log ED,, for noradrenaline (NA) (ordinate) plotted against concentration of Ca++ added to bath fluid (abscissa) for rat portal vein ( O - - O ) and thoracic aorta ( 0 - 0 ) . Points and vertical bars are mean2S.E. from 5 rats. (m) is the mean of the ED,,'s ohtained during the second exposure to 2.5 mM Ca-+-+(see Methods). Reduction of external Ca++ increases ED6, for NA in both tissues.
-8.0
-8.5
-9c
I b
01
0
0 4 08 25 CONC AL3ED Laff (mM1
02
The responses to NA are affected by altering external calcium in both aorta and portal vein but the quantitative aspects differ. Thus, the ED,, for NA is constant in the aorta at concentrations of calcium below 0.1 m M (Fig. 1). In contrast, the portal vein is barely responsive at this concentration of Cat+ (Fig. 2 A) and no ED,,, could be obtained. At higher calcium concentrations ED,, decreases, i.e. the sensitivity to N A increases, in both vessels. It is not likely that the plateau of the ED,,, in the aorta at concentrations of calcium below 0.1 mM is an artefact, since the response to each dose of NA in 0.1 mM Ca"' is much
8
AORTA
b -8
~7
6 5 LOG CONC NA (M)
Fig. 2. Contractile responses (ordinate) produced by several concentrations of noradrenaline (NA) (abscissa) in the presence of reduced external Ca++ in rat portal vein (A) and thoracic aorta (B). The concentrations of Ca++ added to the bath fluid are indicated. The response to each concentration of N A in reduced Ca'+ is expressed a s a per cent of the response to the same concentration of N A in the presence of 2.5 rnM added Ca++. Points and vertical bars are m e a n i S . E . from 5 rats.
268
MORLEY C. SUTTER
larger than in “no added calcium” solutims (Fig. 2 B) so that by inference, the external Ca2+ concentration is less in “no added calcium” than in 0.1 mM Ca2+ solutions. The ED,, for NA can be altered by one or more of 3 factors: 1) altered interaction of NA with adrenergic receptors; 2 ) altered excitation contraction coupling or 3) altered contractile processes per se. There is evidence that severe depletion of tissue calcium affects agonist, but not antagonist-receptor interaction (Tuttle and Moran 1969) but the conditions of our expts. were not nearly so severe as those in which such an effect was observed. Presumably in the present expts. reduced external calcium alters ED,, by interfering with excitation-contraction coupling due to reduction of the amount of calcium available to the contractile elements. In any event the ED,, to NA is altered in both aorta and portal vein by reductions in external calcium concentration. The response of the portal vein to any dose of NA is reduced by lowered external calcium concentration over the ranges tested (Fig. 2 A). In the aorta, however, even at “no added calcium” 25 to 55 per cent of the control (2.5 mM Ca2+)response remains (Fig. 2 B). Hence the magnitude of responses of the portal vein is much more directly dependent on external calcium than those in the aorta. The latter, however, are also affected by increased external calcium. 50% of the maximum response of the aorta to NA is dependent on external Ca++ or at least Ca++in ready equilibrium with the bathing fluid. The virtual complete dependency of the portal vein on external calcium for contraction has been reported previously (for references see Collins et al. 1972). Spontaneous activity in the portal vein was more sensitive to lowered external Ca++ than were responses to NA. Myogenic activity was always absent at 0.4 mM Ca++and was generally present at 0.8 mM Ca++.At this latter concentration the amplitude of spontaneous contractions were very small (approximately 10% of that in 2.5 mM Ca++) whereas contractions induced by NA in 0.8 mM Ca++ (Fig. 2 A) were from 30 to 1000/6 (depending on concentration of NA) of those produced in 2.5 mM Ca++. It is interesting that NA usually induced spontaneous activity even at 0.2 mM Cat+. It is evident that external calcium markedly affects contraction in both rat portal vein and aorta but the quantitative aspects differ. Myogenic activity in the rat portal vein is particularly dependent on external calcium. Relative to the response in normal calcium the residual responses to NA at any level of low external calcium vary with the concentration of NA which indicates a complex interaction between external Ca++ and NA. It seems important that one clearly specifies the external concentration of NA and calcium in a particular experimental situation before attempting to identify sources of calcium which are involved in contraction. This work was supported by the Swedish Medical Research Council Grants 3884 and 14X-0016 and by a Special Travel Fellowship from the British Columbia Heart Foundation.
References COLLINS, G. A., M. C. SUTTER and J. C. TEISER, Calcium and contraction in the rabbit anterior mesenteric portal vein. Canad. J . Physiol. Pharmucol. 1912. 50. 289-299. The effect of calcium depletion on the combination of agonists and TUTTLE,R. R. and N. C. MORAN, competitive antagonists with alpha adrenergic and histaminergic receptors of rabbit aorta. J. Pharmacol. exp. Ther. 1969. 169. 255-263.
Quantitative Effects of External Calcium Concentration on Contraction of Rat Portal Vein Compared to Thoracic Aorta BY
MORLEY C. SUTTER'
The influence of external calcium concentration on the responses to noradrenaline (NA) has been examined in the thoracic aorta and portal vein of the rat. Cumulative concentration-response curves to NA have been produced at each of 6 concentrations of added calcium (ranging from 0 to 2.5 mM Ca2+)and the responses have been compared in two ways: 1) calculation of the ED,, for NA at each concentration of calcium; 2) the response to NA in the altered calcium has been expressed as a percentage of the response to the same concentration of N A in normal (2.5 mM) Ca2+.The results clearly show that alteration of external calcium concentration affects the EDSoand the magnitude of the responses to N A in both the aorta and the portal vein but that there are quantitative differences between the two tissues. The rat portal vein contracts and relaxes spontaneously, hence the effects of the several concentrations of external Ca2+on myogenic activity also could be examined in this tissue. One longitudinal portal vein preparation and one helical strip preparation of the thoracic aorta from each of 5 male Wistar rats (220-265 g) were mounted in organ baths for isometric recording at levels of passive force of 5 and 10 mN respectively. The force signals were electronically integrated. The calcium concentration of the fluid bathing the tissues was varied by adding CaCI, (0-2.5 mM) to a modified Krebs solution of the following composition in mM: NaCl 122; KCI 4.73; NaHCO, 15.5; KH,PO, 1.19; MgCI, 1.19; glucose 11,s; CaNa,-versenate 0.026. It was continuously bubbled with 4 per cent CO, in 0, and kept a t 38°C. The protocol of each experiment was t o allow the tissues to equilibrate in normal Krebs solution for at least 1 h and then to do a cumulative concentration-response curve to NA in concentrations from to M. The tissue was exposed to each concentration of NA for 2 min. After the highest dose of NA the fluid in the bath was changed to one having a different calcium concentration. The sequence was to proceed from fluid containing 2.5 m M added Ca2+ to one with n o added calcium, then to 0.1, 0.2, 0.4, 0.8 and back t o 2.5 m M Ca2+. Responses t o cumulative concentrations of N A were obtained at each level of calcium. Half a n hour was allowed for the tissues to equilibrate in each calcium concentration before doing each concentration-response curve. The response to each concentration of NA was calculated as the mean force developing during the two minute exposure period minus the mean force developed during the control period. Concentration-effect curves for NA a t each level of calcium were plotted as the least squares fit to a true hyperbolic function. The ED,, values in logarithmic form (log ED,,) were used for comparisons. On sabbatical leave from the Department of Pharmacology, University of British Columbia, Vancouver, Canada.
266
267
CALCIUM EFFECT ON PORTAL VEIN
PORTAL VFlN
'\\\
'P
Fig. 1. Log ED,, for noradrenaline (NA) (ordinate) plotted against concentration of Ca++ added to bath fluid (abscissa) for rat portal vein ( O - - O ) and thoracic aorta ( 0 - 0 ) . Points and vertical bars are mean2S.E. from 5 rats. (m) is the mean of the ED,,'s ohtained during the second exposure to 2.5 mM Ca-+-+(see Methods). Reduction of external Ca++ increases ED6, for NA in both tissues.
-8.0
-8.5
-9c
I b
01
0
0 4 08 25 CONC AL3ED Laff (mM1
02
The responses to NA are affected by altering external calcium in both aorta and portal vein but the quantitative aspects differ. Thus, the ED,, for NA is constant in the aorta at concentrations of calcium below 0.1 m M (Fig. 1). In contrast, the portal vein is barely responsive at this concentration of Cat+ (Fig. 2 A) and no ED,,, could be obtained. At higher calcium concentrations ED,, decreases, i.e. the sensitivity to N A increases, in both vessels. It is not likely that the plateau of the ED,,, in the aorta at concentrations of calcium below 0.1 mM is an artefact, since the response to each dose of NA in 0.1 mM Ca"' is much
8
AORTA
b -8
~7
6 5 LOG CONC NA (M)
Fig. 2. Contractile responses (ordinate) produced by several concentrations of noradrenaline (NA) (abscissa) in the presence of reduced external Ca++ in rat portal vein (A) and thoracic aorta (B). The concentrations of Ca++ added to the bath fluid are indicated. The response to each concentration of N A in reduced Ca'+ is expressed a s a per cent of the response to the same concentration of N A in the presence of 2.5 rnM added Ca++. Points and vertical bars are m e a n i S . E . from 5 rats.
268
MORLEY C. SUTTER
larger than in “no added calcium” solutims (Fig. 2 B) so that by inference, the external Ca2+ concentration is less in “no added calcium” than in 0.1 mM Ca2+ solutions. The ED,, for NA can be altered by one or more of 3 factors: 1) altered interaction of NA with adrenergic receptors; 2 ) altered excitation contraction coupling or 3) altered contractile processes per se. There is evidence that severe depletion of tissue calcium affects agonist, but not antagonist-receptor interaction (Tuttle and Moran 1969) but the conditions of our expts. were not nearly so severe as those in which such an effect was observed. Presumably in the present expts. reduced external calcium alters ED,, by interfering with excitation-contraction coupling due to reduction of the amount of calcium available to the contractile elements. In any event the ED,, to NA is altered in both aorta and portal vein by reductions in external calcium concentration. The response of the portal vein to any dose of NA is reduced by lowered external calcium concentration over the ranges tested (Fig. 2 A). In the aorta, however, even at “no added calcium” 25 to 55 per cent of the control (2.5 mM Ca2+)response remains (Fig. 2 B). Hence the magnitude of responses of the portal vein is much more directly dependent on external calcium than those in the aorta. The latter, however, are also affected by increased external calcium. 50% of the maximum response of the aorta to NA is dependent on external Ca++ or at least Ca++in ready equilibrium with the bathing fluid. The virtual complete dependency of the portal vein on external calcium for contraction has been reported previously (for references see Collins et al. 1972). Spontaneous activity in the portal vein was more sensitive to lowered external Ca++ than were responses to NA. Myogenic activity was always absent at 0.4 mM Ca++and was generally present at 0.8 mM Ca++.At this latter concentration the amplitude of spontaneous contractions were very small (approximately 10% of that in 2.5 mM Ca++) whereas contractions induced by NA in 0.8 mM Ca++ (Fig. 2 A) were from 30 to 1000/6 (depending on concentration of NA) of those produced in 2.5 mM Ca++. It is interesting that NA usually induced spontaneous activity even at 0.2 mM Cat+. It is evident that external calcium markedly affects contraction in both rat portal vein and aorta but the quantitative aspects differ. Myogenic activity in the rat portal vein is particularly dependent on external calcium. Relative to the response in normal calcium the residual responses to NA at any level of low external calcium vary with the concentration of NA which indicates a complex interaction between external Ca++ and NA. It seems important that one clearly specifies the external concentration of NA and calcium in a particular experimental situation before attempting to identify sources of calcium which are involved in contraction. This work was supported by the Swedish Medical Research Council Grants 3884 and 14X-0016 and by a Special Travel Fellowship from the British Columbia Heart Foundation.
References COLLINS, G. A., M. C. SUTTER and J. C. TEISER, Calcium and contraction in the rabbit anterior mesenteric portal vein. Canad. J . Physiol. Pharmucol. 1912. 50. 289-299. The effect of calcium depletion on the combination of agonists and TUTTLE,R. R. and N. C. MORAN, competitive antagonists with alpha adrenergic and histaminergic receptors of rabbit aorta. J. Pharmacol. exp. Ther. 1969. 169. 255-263.
