Postsynaptic alpha-adrenoceptor characterization and Ca2+ channel antagonist and activator actions in rat tail arteries from normotensive and hypertensive animals.

Postsynaptic alpha-adrenoceptors in the rat tail artery have been examined by determining the pA2 values for antagonists against several alpha-adrenoceptor agonists. In this tissue the alpha-adrenoceptor agonists all produce concentration-dependent mechanical responses with the following rank order of potency: clonidine greater than norepinephrine greater than phenylephrine greater than UK 14304 greater than B-HT 920. Antagonism by prazosin and yohimbine of phenylephrine, norepinephrine, and clonidine responses does not reveal the anticipated discrimination between alpha 1- and alpha 2-adrenoceptors. Thus, pA2 values for prazosin (9.1-9.5), yohimbine (7.2-7.4), and corynanthine (7.0-7.1) and idazoxan (7.6) do not show large differences between these receptor agonists and suggests the predominance of alpha 1-adrenoceptor mediated contractile responses in this preparation. Significant differences between antagonist activities (pA2 values) in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) artery preparations have not been observed. The sensitivity sequence of alpha-adrenoceptor agonist-induced responses to nifedipine and D 600 is B-HT 920 greater than clonidine greater than phenylephrine greater than norepinephrine. Dependence of agonist response upon extracellular Ca2+ parallels the sensitivity to Ca2+ channel antagonists. Sensitivity to D 600 of phenylephrine responses increased with decreasing concentration of phenylephrine or with receptor blockade by phenoxybenzamine: sensitivity of responses to B-HT 920 was not affected by these procedures. Tail artery strips from WKY and SHR do not exhibit major differences in sensitivity to D 600 or to Ca2+ depletion. Bay k 8644, a Ca2+ channel activator, produces concentration-dependent mechanical responses in the tail artery in the presence of modestly elevated K+ concentrations (10-15 mM): these actions of elevated K+ can be mimicked by both alpha 1- and alpha 2-adrenoceptor agonists including methoxamine, St 587, UK 14304, and clonidine. These studies do not provide clear evidence for the existence of discrete postsynaptic alpha 1- and alpha 2-adrenoceptor populations in rat tail artery as indicated by pA2 values or Ca2+ dependence of response.

Calcium channel activation in vascular smooth muscle by BAY K 8644.

BAY K 8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate) and CGP 28 392 (ethyl-4(2-difluoromethoxyphenyl)-1,4,5,7-tetrahydro-2-methyl-5-++ +oxofuro- [3,4-b]pyridine-3-carboxylate) are closely related in structure to nifedipine and other 1,4-dihydropyridine Ca2+ channel antagonists. However, both BAY K 8644 and CGP 28 392 serve as activators of Ca2+ channels. In the rat tail artery, responses to BAY K 8644 are dependent upon Ca2+ext and prior stimulation by K+ or by the alpha-adrenoceptor agonists, phenylephrine and BHT 920 (6-allyl-2-amino-5,6,7,8,-tetrahydro-4H-thiazolo[4,5-d]azepin dihydrochloride). Responses are blocked noncompetitively by the Ca2+ channel antagonists D-600 [-)-D-600 greater than (+)-D-600) and diltiazem, but competitively by nifedipine (pA2 = 8.27). This suggests that activator and inhibitor 1,4-dihydropyridines interact at the same site. BAY K 8644 potentiates K+ responses and Ca2+ responses in K+-depolarizing media. The leftward shift of the K+ dose--response curve produced by BAY K 8644 suggests that this ligand facilitates the voltage-dependent activation of the Ca2+ channel. The pA2 value for nifedipine antagonism of BAY K 8644 responses is significantly lower than that for nifedipine antagonism of Ca2+ responses in K+ (25-80 mM) depolarizing media (9.4-9.6), suggesting that the state of the channel may differ according to the activating stimulus.

Calcium-dependence and antagonism of responses to alpha 1- and alpha 2-adrenoceptor agonists in vascular tissues from hypertensive and normotensive rats.

Inhibition by D600 (methoxyverapamil) of responses to an alpha 2-adrenoceptor selective agonist, B-HT 920, (6-allyl-2-amino-5, 6, 7, 8-tetrahydro-4H-thiazolo [4, 5-d] azepin dihydrochloride), an alpha 1-adrenoceptor selective agonist, phenylephrine (PE), and a nonselective agonist, noradrenaline (NA), was studied in isolated preparations of the aortae and carotid arteries obtained from young (5-7 weeks) and old (15-17 weeks) hypertensive (SHR) and normotensive (WKY) rats. Maximum responses of WKY tissues to B-HT 920 were the most sensitive, PE-induced responses the least sensitive and maximum responses to NA were intermediate in their sensitivity to inhibition by D600. Sub-maximal responses to NA and PE were not different in their sensitivity to inhibition by D600, but were less sensitive than the responses to B-HT 20. Sub-maximal responses to PE were significantly more sensitive to D600 inhibition than were the maximal responses to this agonist. NA-induced responses of tissues from older SHR were less sensitive to inhibition by D600 when compared to responses in WKY rats. Responses to B-HT 920, in tissues suspended in calcium-free solutions, showed the largest decline compared to NA- and PE-induced responses. We conclude that responses to B-HT 920 largely utilize extracellular calcium. PE- and NA- induced responses mobilize extracellular calcium to varying degrees depending upon the concentration of the agonist employed to elicit the response.

Calcium reactivity and antagonism in portal veins from spontaneously hypertensive and normotensive rats.

Reactivities of portal veins from spontaneously hypertensive rats (SHR) and normotensive controls (Wistar Kyoto, WKY) at 5-7 and 15-17 weeks of age were compared. Systolic blood pressures were not different at 5-7 weeks but those of SHR were significantly elevated (177 +/- 4 mmHg) (1 mmHg = 133.322 Pa) at 15-17 weeks. Spontaneous activity, frequency, and tension were greater in SHR for both age groups. Young SHR were more sensitive to K+ at 5-7 weeks but less sensitive at 15-17 weeks than age-matched WKY rats. Sensitivity to Ca2+ in a K+-depolarizing medium was higher in SHR than in WKY for both age groups. Maximum tension responses to K+ or Ca2+ were greater in SHR. The Ca2+ channel antagonists nifedipine, nitrendipine, and nisoldipine were potent inhibitors of both noradrenaline- and K+-induced responses but did not show differences in inhibitory activity between WKY and SHR.

Inhibition by cinnarizine of the responses of smooth muscle from spontaneously hypertensive and normotensive rats.

A comparison was made of the inhibition by cinnarizine, a calcium antagonist, of the contractile responses of aortic, carotid, and iliac arterial strips and vasa deferentia from 15- to 17-week-old spontaneously hypertensive rats (SHR) and their normotensive counterparts, Wistar Kyoto (WKY) rats. KCl-induced responses of the aorta from both strains of rats and carotid arteries from WKY only were more sensitive to inhibition than were responses to norepinephrine. No significant differences were observed in the inhibition of tissue responses from the two strains of rats with the exception of the K+-induced responses of carotid arterial strips from SHR which were significantly less sensitive to inhibition when compared with carotid strips from WKY.

Calcium antagonists. Some chemical-pharmacologic aspects.

Ca++ serves multiple roles as a regulator and initiator of cellular events. A variety of mechanisms serve to control cellular Ca++ levels and there exists a corresponding diversity of drugs which possess, with varying degrees of potency and selectivity, Ca++-antagonistic properties. Particular interest attaches to a group of agents designated as Ca++-channel or Ca++-entry blockers and includes verapamil, nifedipine, diltiazem, cinnarizine, and prenylamine. These agents function by blockade of the potential dependent Ca++-channel. However, their obvious chemical heterogeneity suggests that several sites and mechanisms of action may exist. A review of some basic questions concerning the action of the Ca++ channel antagonists is presented. The existence of discrete structure-activity relationships is consistent with the concept that these agents have specific sites of action, rather than serving, for example, as, nonspecific membrane-stabilizing agents. This view is further supported by their selectivity of action seen both in tissue selectivity and selectivity of antagonism of agonist responses. Studies of the relationship of these compounds to Ca++ show that they appear to function competitively against Ca++ and to block cellular Ca++ uptake. Moreover, both contractile responses and cellular Ca++ uptake appear equisensitive to these antagonists, providing further evidence that inhibition of Ca++ uptake underlies their inhibition of mechanical response. Of particular importance is the question of selectivity of action. It is quite clear that the Ca++ channel antagonists do not show equal activity toward all Ca++ channels and that major differences in selectivity occur between cardiac and smooth muscle. It is possible that selectivity of antagonism may occur between different vascular beds. Furthermore, Ca++ channels controlling stimulus-secretion coupling appear to be less sensitive to these antagonists than the channels involved in excitation-contraction coupling. Finally, the actions of Ca++ antagonists on non-Ca++-mediated processes, including Na+-dependent and receptor-binding events, are noted. These actions are seen at higher concentrations than those needed to inhibit Ca++ channel events, and their contribution to the therapeutic actions exerted by these antagonists is unknown.

Inhibition by D 600 of norepinephrine- and clonidine-induced responses of the aortae from normotensive (WKY) and spontaneously hypertensive rats (SHR).

Comparison was made of the inhibition by D 600 of the norepinephrine (NE)- and clonidine-induced responses of aortic strips from normotensive (WKY) and spontaneously hypertensive rats (SHR). Clonidine-induced responses of aortae from both strains of rats were markedly more susceptible to inhibition by D 600 than those of NE. Omission of calcium from the physiological solution resulted in a greater reduction in response to clonidine. Aortae from SHR were less susceptible to inhibition by D 600 but showed no difference from WKY rats in their responses in nominally calcium-free physiological solutions. Our results clearly indicate a greater dependence on extracellular calcium by clonidine-induced responses of the aortae relative to those of NE.

Inhibition by methoxyverapamil of the responses of smooth muscle from spontaneously hypertensive and normotensive rats.

Inhibition by methoxyverapamil (D 600) of the responses to norepinephrine (NE) and KCl of vascular (carotid and iliac arteries and aortae) strips and vasa deferentia from spontaneously hypertensive (SHR) and normotensive rats (WKY) were compared. The responses of aortic and carotid arterial strips from SHR to NE and KCl were inhibited to a lesser extent by low concentrations of D 600 (less than 10(-6) M). The isolated preparations of the iliac arteries and the vasa deferentia showed no differences in their susceptibility to inhibition by D 600. The responses to NE and KCl in calcium-free solutions of vascular strips from WKY were generally resistant to inhibition by D 600 (10(-6) M) in contrast to those from SHR which were markedly inhibited by D 600. Our results demonstrate that while the inhibitory action of D 600 on calcium movement may succeed in distinguishing vascular responses of SHR from those of WKY, the inhibitory pattern varies with different tissues and with experimental variations of calcium levels.

Responses to noradrenaline of portal vein strips from normotensive and spontaneously hypertensive rats.

1 Responses to noradrenaline of isolated portal vein strips from 5-7 week and 15-17 week Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) have been examined. 2 15-17 week SHR tissue exhibited a greater sensitivity (lower ED50) to noradrenaline but only at reduced Ca2+EXT levels. 3 pA2 values for the alpha-adrenoreceptor antagonists WB 4101 and prazosin were significantly lower in 5-7 week SHR consistent with some change at the adrenoreceptor level. 4 Veins from SHR relaxed more rapidly in CA2+-free saline following removal of noradrenaline than did veins from WKY. 6 D600 is a competitive antagonist of the Ca2+EXT-dependent component of the noradrenaline response but K1 values were not significantly different in SHR and WKY.

Pharmacology of agents that affect calcium. Agonists and antagonists.

Calcium utilization by smooth muscle is briefly reviewed. A variety of stimulant-evoked calcium mobilization processes exist including utilization of intracellular Ca2+ and translocation of Ca2+ through voltage-sensitive and insensitive channels. The extent of use of these several sources is both stimulant and tissue-dependent and it is probable that most smooth muscle excitation-contraction coupling processes employ more than a single Ca2+ source. A large number of agents have been described as "Ca2+-antagonists" and ideally there would exist antagonists specific for each mobilization process. This does not appear to be the case and the structural diversity of the many antagonists is consistent with multiple modes of action. Prominent among the Ca2+ antagonists are agents as verapamil, D600 and nifedipine which have as one component of their action inhibition of voltage sensitivie Ca2+ channels. However, the mechanisms by which these agents act are unknown and it is also clear that they have important effects at sites other than Ca2+ channels.

Interaction of D 600 with phenoxybenzamine-induced adrenergic blockade.

Pretreatment of the vas deferens with D 600 (1 x 10(-5) M/10 min) prior to addition of phenoxybenzamine (PB) (1 x 10(-7) M/5 min) had no effect on the magnitude of initial adrenergic blockade but significantly increased the rate of recovery from blockade. These results suggest that the long duration of action of PB may be due to its action at calcium mobilization sites.

Spasmolytic action of verapamil, D600 and cinchocaine on the rat vas deferens.

The inhibitory action of verapamil, D600 and a local anesthetic, cinchocaine (CE) was studied on the isolated rat vas deferens. All three drugs inhibited norepinephrine (NE) and KCl dose-response curves in a concentration-dependent manner. Verapamil and D600 were approximately equivalent in their inhibitory effect but were significantly more potent (p < .05) than CE. Cinchocaine enhanced NE dose-response curves prior to inhibition at higher concentrations but did not affect KCl dose-response curves similarly. Inhibition of NE dose-response curves by CE and low concentrations of D600 showed significant negative correlation with the concentration of calcium in the medium. However, inhibition caused by high concentration of D600 (1 X 10(-5) M0 was unaffected by a 10-fold increase in calcium concentration. Contractile responses of the vas deferens to single doses of NE (1 X 10(-4) M) or KCl (40 mM) comprise of an initial fast (F) component and a slow (S) component. The inhibition of the F-component of NE- and KCl-induced responses by D600 and CE was inversely related to the concentration of calcium. Inhibition of the S-component of NE-induced respnses by both drugs was not correlated with calcium levels in contrast to that of KCl-induced responses where the inhibition showed significant negative correlation with the calcium content of the medium. Our results indicate D600 differs from CE in its effects on response to NE but that it is qualitatively similar to CE in its inhibition of KCl-induced responses.

The reactivity of iliac vascular strips from spontaneously hypertensive and normotensive rats.

Comparison of responses to norepinephrine (NE) and KCl of iliac arterial strips from spontaneously hypertensive rats (SHR) and three strains of normotensive rats (NR) were made in tissues obtained from 5- to 7- and 15- to 17-week-old animals. The systolic blood pressures of young rats were essentially similar while the 15- to 17-week-old SHR had markedly higher blood pressures than their normotensive counterparts. The contractility and sensitivity of strips to NE from SHR were less than those from NR in both age groups. Relaxation of NE-induced responses in normal physiological or calcium-free solutions was consistently more rapid with SHR tissues. In calcium-free solution, the fast component of responses to NE was smaller in young SHR; no difference in dependence on calcium was seen in tissues from older rats. Differences in KCl-induced responses were seen only in young animals, where reduced contractility and slower relaxation in normal physiological solutions were obtained with strips from SHR. We conclude that the iliac artery from SHR differs intrinsically from those of NR since the differences in responses were evident in young rats prior to the development of stable hypertensive state in the SHR.

The responses of carotid vascular strips from spontaneously hypertensive and normotensive rats.

The responses to noradrenaline (NA) and KCl of carotid arterial strips from spontaneously hypertensive rats (SHR) and three strains of normotensive rats (NR) were compared using tissue obtained from young (5--7 weeks) or older (15--17 weeks) rats. The maximum responses and sensitivity of strips from SHR were less than those of NR in both age groups. Relaxation of maximum NA-induced responses was consistently faster, and relaxation of KCl-induced responses generally slower with strips from SHR. Carotid strips from young SHR showed a greater dependence on extracellular calcium in their responses to NA and a lesser dependence in their KCl-induced responses than tissues from NR. We conclude that the smooth muscle of carotid arteries from SHR differs intrinsically from that of NR and that the differences in vascular responses may be related to altered excitation-contraction processes in the SHR.

Calcium antagonists and contractile responses in rat vas deferens and guinea pig ileal smooth muscle.

The effect of depletion of extracellular Ca2+ (Ca2+ext) on the loss of responsiveness of the guinea pig ileal longitudinal muscle (g.p.i.l.m.) and the rat vas deferens (r.v.d.) to K+ and cis-2-methyl-4-dimethylaminomethyl-1,3-dioxolane methiodide (CD), and K+ and noradrenaline (NA), has been examined and compared with the effects of a variety of local anesthetics and calcium antagonists. The results indicate that qualitative similarities are apparent with respect to the dependence of agonist-induced activity on Ca2+ext in both the g.p.i.l.m. and r.v.d. Distinct differences, however, in the Ca2+ translocation processes in these two tissues, in response to the different agonists, can be shown by the use of a variety of 'calcium antagonists' thus indicating that such translocation processes are both tissue and agonist selective. It is thus noted that, contrary to the Ca2+ depletion studies, D 600 and the usually more potent BAY-1040 showed no discrimination of action or potency in their ability to inhibit components of the NA response in the r.v.d. In contrast, D 600 and the more potent BAY-1040 selectively inhibited the tonic component of the K+ response. Treatment with SKF 525A and parethoxycaine (PC) in the g.p.i.l.m. and SKF 525A in the r.v.d. resulted in a nonselective inhibition of responses of the tissues to all stimulants. However, in the r.v.d. PC potentiated NA action, and its methobromide (MeBr) derivative potentiated both NA and K+ action and also, like PC, partially shifted to the left the dose-response curve to Ca2+ in NA-depolarizing Ca-free Tyrode's. The quaternary MeBr and the tertiary 2-chloroethyl (2Cl) derivatives of SKF 525A and PC were selectively more effective against CD- than K+ supported contractile activity in the g.p.i.l.m. and the 2Cl derivatives were more effective against NA than K+ responses in the r.v.d. The 2Cl derivative of PC also was more effective in antagonizing the Ca2+ dose-response curve in high-CD or high-NA than in high-K+ Ca2+-free Tyrode's.

The influence of beta-adrenergic antagonists on the adrenergic responses of the rat vas deferens.

The influence of beta-adrenergic antagonists (propranolol, pronethalol, alprenolol, isopropylmethoxamine, H 35/25, sotalol and practolol) on isotonic contractile responses to norepinephrine (NE) was studies. All drugs caused an increase in the maximum responses while depressant effects were seen only with high doses of propranolol, pronethalol and alprenolol. The enhancement of responses to NE was considerably greater at low concentrations of calcium (0.5-1.0 mM) than at high (8 mM) concentrations. The inhibitory effects of propranolol, pronethalol and alprenolol were diminished but not completely overcome by increasing calcium concentrations form 1.8 to 8 mM. Cumulative dose-response curves of calcium showed no increase in maximum responses although responses to low concentrations of calcium were augmented by sotalol and practolol. Evidence suggests that the enhancing effects of these drugs may be due to their facilitatory effect on calcium mobilization following alpha-adrenoceptor activation while their depressant properties probably reflect their membrane stabilizing properties.

Persistent enhancement of potassium-induced responses of the rat vas deferens by desipramine.

The effect of desipramine on the cumulative dose-response curves of noradrenaline and potassium (K+) was examined on the isolated rat vas deferens. An exposure of 10 min to 10(-7) M desipramine caused a leftward shift and an increase in the maximum response of cumulative dose-response curves of noradrenaline. Desipramine (10(-7) M), in contact with the tissue for 10 min, enhanced responses to cumulative additions of K+ without causing a consistent change in threshold concentrations. Wash-out of desipramine resulted in a rapid loss of enhanced maximum response to noradrenaline while the maximum response to K+ did not show any decrease for up to 120 min after wash-out of drug. One possible explanation for the persistent enhancement of K+-induced responses may be that desipramine causes postjuntional changes which selectively influence contractile responses of this tissue to K+.

The effects of lanthanum and thulium on the mechanical responses of rat vas deferens.

1. The contractile responses of rat vas deferens to noradrenaline and K+ are composed of phasic and tonic components both of which are dependent upon the concentration of extracellular Ca2+. 2. Lanthanum, La3+, and thulium ions, Tm3+, inhibited the noradrenaline and K+ induced responses, complete inhibition being obtained at approximately 10(-3) M-Ln3+. 3. La3+ and Tm3+ were equally effective in inhibiting noradrenaline and K+ responses. The phasic and tonic components of the noradrenaline response were equally sensitive to lanthanide cations, Ln3+, but the phasic component of the K+ response was more sensitive than the tonic component. 4. 170Tm binding did not show any saturable component over the concentration range in which inhibition of the pharmacological response was obtained. 5. It is suggested that the actions of Ln3+ in the rat vas deferens are mediated through some kind of membrane stabilization rather than via a specific Ca2+ binding site concerned with excitation-contraction coupling, the mechanism previously postulated for the Ln3+ action in guinea-pig ileal longitudinal muscle.

The action of the ionophores, X-537A and A-23187, on smooth muscle.

The cation ionophore X-537A in the concentration range of 10(-6) to 3 X 10(-5) M produced contractions in the rat and guinea-pig vas deferens. No contractile effect was produced in either of the vasa deferentia preparations by the ionophore A-23187 in the concentration range of 10(-7) to 5 X 10(-5) M. In contrast, X-537A had no contractile effect on the guinea-pig ileal longitudinal smooth muscle while A-23187 produced a dose and [Ca2+] dependent contraction. The contractile effect of X-537A in the vasa deferentia preparations is abolished by phenoxybenzamine or prior reserpine treatment and is therefore attributed to the release of norepinephrine. The effect of A-23187 in the intestinal smooth muslce is attributed to a direct Ca2+ transporting action since its contractile effect is unaffected by histamine, acetylcholine, or 5-hydroxytryptamine antagonists.