The store-operated Ca2+ entry (SOCE) pathway has aroused much interest recently not only because of its unusual nature as retrograde signaling, but also due to its wide occurrence and its possible role in physiological and pathophysiological situations. A number of synthetic or naturally occurring drugs recently used to block this Ca2+ entry pathway are briefly reviewed. Although important and interesting information has been obtained using these putative SOCE blockers described in this review, they indeed have sites of action other than the SOCE channels, and caution must be exercised in using them as putative tools to study SOCE. For instance, the highly variable potency of some synthetic blockers (SK&F 96365 and LOE 908) to inhibit SOCE has provided indirect evidence for the heterogeneous nature of the SOCE channels, an observation consistent with the differential Mn2+ permeability through SOCE in various cell types. The use of SK&F 96365 at relatively high concentrations has unexpectedly revealed its potential as an opener of a novel cation entry pathway. The ability of LU52396 to discriminate the SOCE channel in its closed/open states may be useful in the analysis of the kinetics of SOCE channel activation/inactivation. The possible presence of both agonistic and antagonistic saponins derived from ginseng plants for the study of SOCE deserves more rigorous experimental investigations, which may lay new ground for the development of new types of Ca2+ antagonists (and/or agonists) from the natural resources.
There are two known major angiotensin II receptor subtypes, type 1 (AT1) and type 2 (AT2), both of which are present in the brain. AT1 and AT2 receptors occur in characteristic distributions that are highly correlated with the distribution of angiotensin II-like immunoreactivity in nerve terminals. Acting through the AT1 receptor in the central nervous system, angiotensin II has effects on fluid and electrolyte homeostasis, neuroendocrine systems, autonomic pathways regulating cardiovascular function and behavior. While the role of the AT2 receptor in the brain is less well understood, recent knockout studies point to their involvement in behavioral and cardiovascular functions. We discuss here evidence regarding the function of the AT2 receptor in the brain, determined using mice lacking the AT2 receptor.
Recently discovered endomorphin-1 and -2 are the first endogenous agonists selective for the μ-opioid receptor. We examined the antinociceptive effect and enzymatic degradation of endomorphin-1 in the newborn rat spinal cord. Endomorphin-1 inhibited the binding of [3H][D-Ala2, N-Me-Phe4, Gly-ol5] enkephalin (DAMGO) to the membrane fraction of the newborn rat spinal cord as potently as DAMGO and morphine. Endomorphin-1 at 1 - 1, 000 nM reduced the slow ventral root potential, which reflects noxious transmission in the isolated newborn rat spinal cord, concentration-dependently via the μ-opioid receptor. A similar effect was observed with endomorphin-2. The newborn rat spinal cord homogenate degraded endomorphin-1 in a 120-min incubation procedure, while it degraded [Leu5]enkephalin even in a 30-min incubation procedure. The degradation of endomorphin-1 was inhibited by actinonin but not by thiorphan. These results showed that in the newborn rat spinal cord, endomorphins had high affinity for the μ-opioid receptor and exerted μ-opioid-receptor - mediated inhibitory effects on noxious responses. Endomorphin-1 was degraded by peptidases, but slowly compared with [Leu5]enkephalin degradation, and the degrading enzymes were actinonin-sensitive peptidases.
α1-Adrenergic receptors (α1-ARs) play critical roles in the regulation of a variety of physiological processes. Increasing evidence suggests that multiple receptor subtypes of α1-ARs regulate these physiological processes. Molecular cloning has identified three distinct cDNAs encoding α1-AR subtypes (α1A, α1B and α1D) that are structurally homologous. Among the α1-AR subtypes, the function of the α1D-AR remains unclear. In order to examine the physiological role of a1D-AR, we cloned and characterized a gene for the mouse α1D-AR. Using a mouse α1D-AR cDNA as a probe, we isolated the gene for the mouse α1D-AR from a mouse genomic library. The α1D-AR consists of two exons and an intron that interrupts the coding region of the putative sixth transmembrane domain. The 5′-flanking region of exon 1 contains neither a TATA box nor a CAAT box but is high in GC content and contains several Sp1 binding sites (GC boxes). This pattern is similar to promoters described for other members of α1-ARs. The untranslated region also contains putative cyclic AMP response elements. Isolation of this gene will allow further investigation, via gene knock-outs and deletion mutants, of the mechanisms of transcriptional regulation and a greater understanding of the physiological role of α1D-AR.
We studied the effect of chronic nitric oxide synthase (NOS) blockade in the brain on mean arterial pressure [MAP (mmHg)], heart rate [HR (bpm)] and baroreceptor reflex sensitivity [BRS (mean slope: bpm/mmHg)] in Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Intracerebroventricular (i.c.v.) infusion of the nonselective NOS inhibitor N-Nitro-L-arginine-methylester (LNAME) (50 μg/kg per day, 11 - 12 days) increased MAP in WKY and SHR (125±2.1 vs 118±1.1 controls, P<0.01 and 179±3.59 vs 156±4.0 controls, P<0.001, respectively) without affecting HR. In L-NAME-treated WKY, BRS to bradycardia was suppressed (-0.79±0.09 vs -1.76±0.17 controls, P=0.001), whereas in SHR, L-NAME did not affect BRS to bradycardia. BRS to tachycardia remained unaffected in either strain. In WKY, 7-nitroindazole (7-NI·Na+) (34 μg i.c.v./kg per day, 11 - 12 days), a selective nNOS inhibitor, did not affect MAP or HR, but BRS to bradycardia and tachycardia was decreased (-0.37±0.20 vs -0.97±0.41 controls, P<0.01 and -1.78±0.20 vs -2.52±0.40 controls, P=0.05, respectively). In SHR, the same dose of 7-NI·Na+ increased resting MAP (171±5.00 vs 150±7.00 controls, P<0.05) without affecting HR or BRS to bradycardia or tachycardia. Thus in WKY, BRS to acute changes in systemic blood pressure (BP) is regulated by NO produced by nNOS in the brain, serving as a neurotransmitter in sympathetic and parasympathetic efferent pathways. In SHR, systemic BP is regulated in part by NO released by the type I NOS isoenzyme in the brain.
The effect of nipradilol on the isoproterelol-induced depression of contractions of the soleus muscle of the anesthetized cats was studied. Isoproterenol (0.3 μg/kg) injected intravenously decreased the tension and degree of fusion of incomplete tetanic contractions of the soleus muscle of the anesthetized cats. The effect of isoproterenol was blocked by nipradilol (≥3 μg/kg), desnitro-nipradilol (≥10 μg/kg) and propranolol (≥10 μg/kg), but not by nitroglycerin (10 - 100 μg/kg). Nipradilol (30 μg/kg) and desnitro-nipradilol (300 μg/kg) almost completely antagonized the depressor effects of isoproterenol. These results coupled with evidence that nipradilol does not penetrate the blood-brain barrier indicate that nipradilol exerts an anti-tremor action by blocking peripheral β2-adrenoceptors.
The gastrokinetic activity of SK-951 ((-)4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro-2, 3-dihydro-2-methylbenzo[b]furan-7-carboxamide hemifumarate), a benzofuran derivative with 5-hydroxytryptamine (5-HT)4-receptor agonist activity, was studied in rats and dogs. The effects of SK-951 were also investigated in a model of vagotomy-induced gastroparesis in comparison with cisapride. In rats, both SK-951 and cisapride enhanced gastric emptying of liquids (phenol red) at a dose of 1 - 100 mg/kg, p.o. Gastric emptying of liquid (acetaminophen) in fasted beagle dogs was enhanced significantly by SK-951 (1.0 mg/kg, i.v.), whereas the effect of cisapride (0.2 - 1.0 mg/kg, i.v.) was not statically significant. Similar results were found when radiopaque markers were given with standard meal to dogs with vagotomy-induced gastroparesis. The delayed gastric emptying of radiopaque markers by vagotomy was reversed by SK-951 (1.0 mg/kg, i.v.), whereas cisapride showed no effect at doses from 0.1 to 1.0 mg/kg, i.v. These results indicated that oral and intravenous administration of SK-951 accelerates gastric emptying of both liquids and solids in animal models. Thus, SK-951 may be a highly potent and useful prokinetic agent in comparison to cisapride.
We have previously found that human chymase selectively cleaves big endothelins (ETs) at the Tyr31-Gly32 bond to produce 31-amino-acid endothelins, ETs (1 - 31). In the present study, we investigated the effects of ETs (1 - 31) on changes in intracellular free Ca2+ ([Ca2+]i) in cultured human coronary artery smooth muscle cells (HCASMCs) using confocal laser microscopy. ETs (1 - 31) increased [Ca2+]i in a concentration-dependent manner. Phosphoramidon did not inhibit the increases in [Ca2+]i caused by ETs (1 - 31). The [Ca2+]i increases induced by ETs (1 - 31) were compared to those of ETs (1 - 21) and big ETs. ET-1 (1 - 21) was about 10-times more potent than big ET-1 or ET-1 (1 - 31), whereas big ET-2 was 10-times less potent than ET-2 (1 - 21) or ET-2 (1 - 31). ETs (1 - 31) may induce [Ca2+]i increase through ETA-type or ETA-type-like receptors. The 10-12 M ET (1 - 31)-induced increases in [Ca2+]i were not affected by removal of extracellular Ca2+, but were inhibited by thapsigargin. These results suggested that ET-1, -2 and -3 (1 - 31) showed similar potencies in increasing [Ca2+]i and mechanisms of ET (1 - 31)-induced increases in [Ca2+]i may be similar among the three ETs (1 - 31).
We determined the effects of toluene exposure on activation or inhibition of adenylyl cyclase by stimulating human β2-adrenergic receptors (β2-AR) and muscarinic acetylcholine receptor (mAChR) m2 subtypes, respectively, expressed in CHO cells. The formation of cAMP via β2-AR stimulation was slightly but not significantly facilitated in the presence of 3.7 μM toluene. On the other hand, the inhibition of adenylyl cyclase by 10 μM of carbamylcholine stimulation of mAChR m2 subtypes was attenuated in the presence of toluene. These results strongly suggest that toluene affects activation of Gi rather than Gs.
Arginine vasopressin (AVP) (0.03 - 3 μM) produced transient negative responses that were followed by slowly developing positive inotropic responses in guinea pig papillary muscles. The inotropic responses were antagonized by OPC-21268 (10 μM), but not by OPC-31260, and suppressed by neomycin (3 mM). The negative inotropic response was hardly affected by staurosporine (10 nM) and H-7 (10 μM). AVP (1 μM) elicited a transient decrease followed by a slowly developing increase in ICa. The latter increase in ICa was not elicited in the presence of staurosporine (1 nM). These results indicate that AVP stimulates V1 receptors that couple to phosphoinositide hydrolysis and thereby increases intracellular Ca2+ and activates protein kinase C.
We examined the effect of N-methyltyramine (NMT) on α2-adrenoceptor. NMT (10-8 - 10-3 M) inhibited the binding of [3H]p-aminoclonidine to α2-adrenoceptor dose-dependently. However, the IC50 value for NMT (5.53×10-6 M) was higher than that for RX821002, an α2-adrenoceptor antagonist (1.07×10-8 M). RX821002 (5 mg/kg, i.p.) inhibited hypermotility induced by scopolamine (8 mg/kg, s.c.) in male ddY mice. NMT (20 or 100 mg/kg, i.p.) was found to have a dose-dependent inhibitory effect similar to that of RX821002. These findings indicate that NMT has the properties of an α2-adrenoceptor antagonist. However, the affinity of NMT for α2-adrenoceptor is weaker than that of RX821002.