C. Cauvin

Differences in norepinephrine activation and diltiazem inhibition of calcium channels in isolated rabbit aorta and mesenteric resistance vessels.

The mechanisms of norepinephrine stimulation of calcium ion entry in isolated rabbit aorta and mesenteric resistance vessels were studied through measurements of effects on calcium-45 influx, tension, and membrane potential. The resistance vessels were considerably less sensitive to norepinephrine than the aorta. The aorta exhibited complex dose-response curves for norepinephrine-stimulated calcium influx and contraction, whereas these were simple in the arterioles. Both vessels were depolarized with increasing concentrations of potassium. Norepinephrine did not depolarize the aorta, whereas it did depolarize the mesenteric resistance vessels. This result supports the contention that norepinephrine opens receptor-operated channels to induce calcium entry in the aorta, while it may activate potential sensitive calcium channels in the mesenteric resistance vessels. However, the maximum depolarization with norepinephrine (10−4 m) in the arterioles was completely blocked by 10−5 m diltiazem, whereas that induced by 80 Mm potassium was unaltered by the diltiazem. Furthermore, 10−4 m norepinephrine was able to stimulate virtually the same contraction and calcium influx in 80 mM potassium-depolarized arterioles as in normal polarized tissues. These results are consistent with norepinephrine opening of receptor-operated channels to allow calcium entry in the rabbit mesenteric resistance vessels. That the behavior of norepinephrine-activated channels in the aorta is more complex than in the arterioles is further illustrated by a dramatically decreasing sensitivity of norepinephrine-stimulated calcium influx to diltiazem with increasing norepinephrine in the aorta but not in the arterioles. We have hypothesized that the complexity of the norepinephrine-stimulated calcium influx in the aorta compared to the resistance vessels may be related to the substantial release of intracellular calcium in the former, such that, as release of intracellular calcium is increased, sensitivity of norepinephrine-activated channels to diltiazem decreases.

Extracellular Ca2+ dependence and diltiazem inhibition of contraction in rabbit conduit arteries and mesenteric resistance vessels.

The dependence of norepinephrine-(NE-) and high potassium (80 mM K) depolarization-induced contractions on extracellular versus intracellular calcium (Ca2+) pools was studied in strips of rabbit superior mesenteric artery and its branches, and in perfused mesenteric resistance vessels. 80 mM K contractions were abolished in the presence of 0 Ca2+ 2 mM EGTA in all arteries studied, whereas 10(-5) M NE-induced contractions and stimulated 45Ca efflux in 0 Ca2+ 2 mM EGTA decreased in a graded fashion from proximal to distal arteries. These data indicate a decreasing release of intracellular Ca2+ and an increasing dependence on extracellular Ca2+ for NE-induced contractions as one proceeds from proximal to distal arteries. This pattern was paralleled by increasing sensitivity of NE-induced contractions to inhibition by diltiazem (10(-9)-10(-4) M) from proximal to distal arteries, while inhibition of 80 mM K contractions was similar in all vessels. 45Ca influx induced by 10(-5) M NE in the resistance vessels, wherein the NE does not release intracellular Ca2+, is approximately 10,000-fold more sensitive to the action of diltiazem than that in the aorta. However, when the aorta is activated by 10(-8) M NE, it becomes more sensitive to inhibition by diltiazem than when it is activated by 10(-5) M NE. The former NE concentration does not release intracellular Ca2+ in the aorta, whereas the latter does. Thus, it appears that NE-induced 45Ca influx is most susceptible to inhibition by diltiazem when the NE has not also released intracellular Ca2+. We suggest that the release of intracellular Ca2+ by NE may make its stimulated Ca2+ influx less susceptible to inhibition by diltiazem.

Effects of Ca antagonists on Ca fluxes in resistance vessels

We have examined contractions and 45Ca fluxes induced by norepinephrine (NE) and 80 mM potassium (high K) depolarization and their inhibition by diltiazem in rabbit mesenteric resistance vessels. Contraction induced by both NE and high K depended almost completely on extracellular Ca. Dose-response curves for diltiazem inhibition of NE (10(-5) M) and high K contractions showed ED50 values of 1 X 10(-8) and 6 X 10(-7) M, respectively, indicating that the receptor-operated channel (ROC) was more sensitive than the potential-operated channel (POC) to the action of diltiazem. Diltiazem (10(-6) M) was shown to inhibit NE- and 80 mM K-stimulated 45Ca influx effectively by 87 +/- 15 and 85 +/- 10%, respectively. Comparison of these data to those obtained from aorta suggest that although the sensitivity of the POC is approximately the same in aorta and mesenteric resistance vessels, the sensitivity of the ROC is much greater in the latter. This increased sensitivity is paralleled by a greatly decreased role of intracellular Ca release in NE contraction in mesenteric resistance vessels.

Effects of dihydropyridines on tension and calcium-45 influx in isolated mesenteric resistance vessels from spontaneously hypertensive and normotensive rats

Contractile tension responses to norepinephrine and depolarizing potassium (80 mM K+), as well as calcium-45 influx stimulated by these agents, were studied in isolated mesenteric resistance vessels (each 100 microM internal diameter) from spontaneously hypertensive rats (SHRs) and from normotensive Wistar Kyoto rats (WKYs). Inhibitory effects of 2 dihydropyridine Ca++ antagonists, PN 200-110 (isradipine) and nisoldipine, on these parameters were also determined. Contractile responses to 80 mM K+ were inhibited by both Ca++ antagonists with the same potency and efficacy in SHR compared with WKY vessels (PN 200-110 IC50 = 2.8 +/- 1.3 X 10(-8) M in SHRs and 2.5 +/- 1.5 X 10(-8) M in WKYs; nisoldipine IC50 = 1.1 +/- 0.4 X 10(-8) M in SHRs and 1.2 +/- 0.9 X 10(-8) M in WKYs). However, contractile responses to norepinephrine (10(-4) M) were inhibited less potently by nisoldipine in SHR vessels (IC50 = 2.2 +/- 0.3 X 10(-9) M) compared with WKY vessels (IC50 = 1.6 +/- 0.6 X 10(-10) M). Similarly, PN 200-110 tended to be less (but not significantly less) potent in SHR vessels (IC50 = 3.3 +/- 1.8 X 10(-8) M) than in WKY vessels (IC50 = 3.4 +/- 0.9 X 10(-9) M); its efficacy was significantly depressed in the SHR vessels (by approximately 20%). When norepinephrine-stimulated calcium-45 influx was determined in the presence of these Ca++ antagonists, a similar profile emerged with respect to a comparison of SHR and WKY vessels. These results support a previously hypothesized alteration in receptor-activated Ca++ influx pathways in SHR mesenteric resistance vessels.

The Effects of Ca2+ Antagonists on Isolated Rat and Rabbit Mesenteric Resistance Vesselsa

The phenomena that underlie the selective effects of organic Ca2+ antagonists in inhibiting vascular smooth muscle preferentially over other tissues and in selectively inhibiting certain vascular smooth muscles and certain modes of activation over others have been extensively studied and reviewed.14 Although considerable progress has been made in the characterization of the voltage dependence of Ca2+ antagonist potency in Ca2+ channel blockade,’-’ and of use or frequency dependence of Ca2+ antagonist blockade,1v638 many observations relating to Caz+ antagonist selectivity remain unexplained. We present in this paper a summary of the observations we have made over the last five years in the study of the effects of CaZ+ antagonists on peripheral resistance vessels from the mesenteric vasculature of rabbit and rat. We will attempt to relate these observations to those of others, who are approaching the questions pertaining to Ca2+ antagonist selectivity through the study of Ca2+ currents in single cells or single Ca2+ channels. Finally, we will reiterate a hypothesis, which remains to be directly tested, that the Ca2+ antagonist sensitivity of agonist-activated Ca2+ entry into vascular smooth muscle depends on the extent to which the agonist mobilizes intracellular Ca2+.2.9-11

Vascular Smooth Muscle Calcium Channels

SUMMARY Vascular smooth muscle Ca channels function in excitation-contraction coupling. A survey of recent literature reveals several types of excitable Ca channels. There are at least two plasmalemmal Ca channels that are primarily activated by depolarization. In addition, there also exists evidence for the presence of Ca channels in conduit arteries that are primarily activated by agonists. Under circumstances of compromised sarcoplasmic reticulum (SR) Ca accumulation, Ca that enters through the nonregulated Ca leak also contributes to tension development. The Ca release from the SR appears to be mediated by large Ca channels that are activated by free Ca, inositol-1,4,5-trisphosphate, and free ATP. The differential sensitivity to procaine suggests the presence of two separate excitable Ca channels in vascular smooth muscle SR in addition to a basal Ca leak. This presentation concludes with a theoretical model describing how vascular smooth muscle Ca metabolism may be altered in hypertension and how a Ca antagonist may lead to reduction of blood pressure.

Effects of Ca2+ Antagonists on Isolated Rabbit Mesenteric Resistance Vessels as Compared to Rabbit Aorta

We have reported (Cauvin et al. 1984 a) that norepinephrine-(NE-)activated rabbit mesenteric resistance vessels are far more sensitive to inhibition by diltiazem than are aortae. Not only are the contractions far more sensitive to diltiazem, but also the NE-stimulated 45Ca2+ influx. Activation with 80 mM K+, on the other hand, is similar in its diltiazem sensitivity in the two vessel types. We have now extended our studies to include three other Ca2+ antagonists (CAts): D600, and the two dihydropyridines, nisoldipine and PY108-086. We have hypothesized previously (Cauvin et al. 1984 a) that the high sensitivity of the NE-activated mesenteric resistance vessels to diltiazem may be related to their relative lack of agonistinduced release of intracellular Ca2+ . We have attempted to further investigate this possibility and to study other possible theoretical reasons why Ca2+ influx stimulated by NE in the rabbit mesenteric resistance vessels is more sensitive to CAts than is that in the aorta.

Membrane Ca2+ Permeability and Calcium Antagonistic Effects in Resistance Vessels of Spontaneously Hypertensive Rats

This study compares resting and stimulated 45Ca entry in vitro in mesenteric resistance vessels (MRV) of Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). The stimulating agent used was norepinephrine (NE). The results show clearly that Ca2+ influx into smooth muscle cells from resistance vessels was much higher in the SHR than in the WKY rats. Potential sensitive Ca2+ channels (PSC) and receptor-operated Ca2+ channels (ROC) may possibly be involved, although there is as yet no evidence for the existence of ROC in MRV in the rat. Further investigations are now needed to establish whether the enhanced stimulated Ca2 + entry is related causally to increased peripheral resistance, i.e., whether it occurs in vivo and what temporal relation it has to the onset of hypertension. Mechanistically, it remains to be established which aspect of channel function is involved in the enhanced Ca2 + entry, and how this is connected with cation stabilization of smooth muscle plasmalemmae in hypertensive individuals.