Nonselective Calcium Channel Blocker Research Articles

Multiple sources of internal calcium stores mediate ethanol-induced presynaptic inhibitory GABA release in the central nucleus of the amygdala in mice.

Ethanol can enhance GABA release in various brain regions via presynaptic mechanisms. However, the presynaptic action of ethanol on inhibitory GABA release is still not well understood. Since calcium is required for neurotransmitter release from presynaptic terminals, the purpose of this study was to investigate the role of both internal and external calcium signaling in ethanol-induced enhancement of GABA release within the central amygdala nucleus (CeA) in acute brain slice preparations. Whole-cell patch clamp electrophysiology was used to record miniature GABAA receptor-mediated inhibitory postsynaptic currents (mIPSCs) from CeA neurons. Ethanol-enhanced mIPSCs were recorded in the presence of antagonists that regulate internal and external calcium-mediated processes. Bath-applied ethanol dose-dependently increased the mean frequency of mIPSCs without altering mIPSC amplitude. Ethanol-induced increases in mIPSC frequency were antagonized by dantrolene, 2-APB, and the endoplasmic reticulum calcium pump (SERCA) antagonists thapsigargin and cyclopiazonic acid (CPA). Blocking calcium release from mitochondria or via exocytosis with ruthenium red also attenuated mIPSCs while frequency was not altered in the presence of a non-selective calcium channel blocker cadmium. The L-type calcium blocker nifedipine, but not its analogue nimodipine, blocked ethanol-induced enhancement in CeA neurons. These results demonstrate ethanol-induced presynaptic release of GABA is mediated by internal calcium stores and by disrupting neurotransmitter exocytosis within the CeA, a critical brain area involved in drugs of abuse and alcohol addiction.

Polymodal Transient Receptor Potential Vanilloid (TRPV) Ion Channels in Chondrogenic Cells.

Mature and developing chondrocytes exist in a microenvironment where mechanical load, changes of temperature, osmolarity and acidic pH may influence cellular metabolism. Polymodal Transient Receptor Potential Vanilloid (TRPV) receptors are environmental sensors mediating responses through activation of linked intracellular signalling pathways. In chondrogenic high density cultures established from limb buds of chicken and mouse embryos, we identified TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6 mRNA expression with RT-PCR. In both cultures, a switch in the expression pattern of TRPVs was observed during cartilage formation. The inhibition of TRPVs with the non-selective calcium channel blocker ruthenium red diminished chondrogenesis and caused significant inhibition of proliferation. Incubating cell cultures at 41 °C elevated the expression of TRPV1, and increased cartilage matrix production. When chondrogenic cells were exposed to mechanical load at the time of their differentiation into matrix producing chondrocytes, we detected increased mRNA levels of TRPV3. Our results demonstrate that developing chondrocytes express a full palette of TRPV channels and the switch in the expression pattern suggests differentiation stage-dependent roles of TRPVs during cartilage formation. As TRPV1 and TRPV3 expression was altered by thermal and mechanical stimuli, respectively, these are candidate channels that contribute to the transduction of environmental stimuli in chondrogenic cells.

Investigation of the role of non-selective calcium channel blocker (flunarizine) on cerebral ischemic–reperfusion associated cognitive dysfunction in aged mice

The present study was designed to investigate the role of flunarizine (a non-selective calcium channel blocker) on cerebral ischemic–reperfusion associated cognitive dysfunction in aged mice. Bilateral carotid artery occlusion of 12min followed by reperfusion for 24h was given to induce cerebral injury in male Swiss mice. The assessment of learning & memory was performed by Morris water maze test; motor in-coordination was evaluated by rota rod, lateral push and inclined beam walking tests; cerebral infarct size was quantified by triphenyltetrazolium chloride staining. In addition, reduced glutathione (GSH), total calcium and acetylcholinesterase (AChE) activity were also estimated in aged brain tissue. Donepezil treated group served as a positive control in this study. Ischemia reperfusion (I/R) injury produced significant increase in cerebral infarct size. A significant loss of memory along with impairment of motor performance was also noted. Further, I/R injury also produced significant increase in levels of total calcium, AChE activity and decrease in GSH levels. Pretreatment of flunarizine significantly attenuated I/R induced infarct size, behavioral and biochemical changes. Hence, it may be concluded that, a non-selective calcium channel blocker can be useful in I/R associated cognitive dysfunction due to its anti-oxidant, anti-infarct and modulatory actions of neurotransmitters & calcium channels.

Non-Selective Calcium Channel Blocker Bepridil Decreases Secondary Pathology in Mice after Photothrombotic Cortical Lesion

Experimental studies have identified a complex link between neurodegeneration, β-amyloid (Aβ) and calcium homeostasis. Here we asked whether early phase β-amyloid pathology in transgenic hAPPSL mice exaggerates the ischemic lesion and remote secondary pathology in the thalamus, and whether a non-selective calcium channel blocker reduces these pathologies. Transgenic hAPPSL (n = 33) and non-transgenic (n = 30) male mice (4–5 months) were subjected to unilateral cortical photothrombosis and treated with the non-selective calcium channel blocker bepridil (50 mg/kg, p.o., once a day) or vehicle for 28 days, starting administration 2 days after the operation. Animals were then perfused for histological analysis of infarct size, Aβ and calcium accumulation in the thalamus. Cortical photothrombosis resulted in a small infarct, which was associated with atypical Aβ and calcium accumulation in the ipsilateral thalamus. Transgenic mice had significantly smaller infarct volumes than non-transgenic littermates (P<0.05) and ischemia-induced rodent Aβ accumulation in the thalamus was lower in transgenic mice compared to non-transgenic mice (P<0.01). Bepridil decreased calcium load in the thalamus (P<0.01). The present data suggest less pronounced primary and secondary pathology in hAPPSL transgenic mice after ischemic cortical injury. Bepridil particularly decreased calcium pathology in the thalamus following ischemia.

Bepridil decreases Aβ and calcium levels in the thalamus after middle cerebral artery occlusion in rats.

Alzheimer's disease (AD) and cerebral ischaemia share similar features in terms of altered amyloid precursor protein (APP) processing and β-amyloid (Aβ) accumulation. We have previously shown that Aβ and calcium deposition, and β-secretase activity, are robustly increased in the ipsilateral thalamus after transient middle cerebral artery occlusion (MCAO) in rats. Here, we investigated whether the non-selective calcium channel blocker bepridil, which also inhibits β-secretase cleavage of APP, affects thalamic accumulation of Aβ and calcium and in turn influences functional recovery in rats subjected to MCAO. A 27-day bepridil treatment (50 mg/kg, p.o.) initiated 2 days after MCAO significantly decreased the levels of soluble Aβ40, Aβ42 and calcium in the ipsilateral thalamus, as compared with vehicle-treated MCAO rats. Expression of seladin-1/DHCR24 protein, which is a potential protective factor against neuronal damage, was decreased at both mRNA and protein levels in the ipsilateral thalamus of MCAO rats. Conversely, bepridil treatment restored seladin-1/DHCR24 expression in the ipsilateral thalamus. Bepridil treatment did not significantly affect heme oxygenase-1- or NAD(P)H quinone oxidoreductase-1-mediated oxidative stress or inflammatory responses in the ipsilateral thalamus of MCAO rats. Finally, bepridil treatment mitigated MCAO-induced alterations in APP processing in the ipsilateral thalamus and improved contralateral forelimb use in MCAO rats. These findings suggest that bepridil is a plausible therapeutic candidate in AD or stroke owing to its multifunctional role in key cellular events that are relevant for the pathogenesis of these diseases.

Efficacy and Tolerability of Topical 0.05% Flunarizine in Patients With Open-angle Glaucoma or Ocular Hypertension—A Pilot Study

To evaluate the efficacy and tolerability of flunarizine, a nonselective calcium channel blocker, on intraocular pressure (IOP) in patients with open-angle glaucoma (OAG) or ocular hypertension (OH). Randomized, placebo-controlled, double blind, cross-over phase II trial. Ten patients diagnosed with bilateral OAG or OH were included. Each patient served as his own control. On the first study visit, 1 drop of either 0.05% flunarizine or placebo was applied in the study eye. Patients were followed-up during 6 hours (0, 15, 30, and 60 min, 2, 3, 4, and 6 h) for vital signs, slit lamp examination, corneal fluorescein staining, and IOP. After a wash-out period of 4 weeks, 1 drop of either placebo or flunarizine was applied in the same eye and patients were followed-up for another 6 hours. The flunarizine group showed a statistically significant decrease from baseline at T2, T3, T4, and T6 hours (P=0.01, P=0.03, P=0.01, and P=0.03, respectively). No significant difference in IOP decrease from baseline (P>0.06) was observed between the 2 groups. There was no statistically significant difference in all of the following parameters: limbal hyperemia, bulbar or palpebral conjunctival hyperemia, corneal staining with fluorescein, best-corrected visual acuity, comfort of eye drops, and vital signs. The absence of a statistically significant difference in IOP reduction between the 2 groups may be due to the small number of patients included. However, a single dose of 0.05% flunarizine significantly reduced IOP when compared with baseline values, whereas this was not observed with placebo.

P22 Clinical and pathological heterogeneity in partial merosin deficiency

P22 Clinical and pathological heterogeneity in partial merosin deficiency

N-Palmitoyl Glycine, a Novel Endogenous Lipid That Acts As a Modulator of Calcium Influx and Nitric Oxide Production in Sensory Neurons

N-arachidonoyl glycine is an endogenous arachidonoyl amide that activates the orphan G protein-coupled receptor (GPCR) GPR18 in a pertussis toxin (PTX)-sensitive manner and produces antinociceptive and antiinflammatory effects. It is produced by direct conjugation of arachidonic acid to glycine and by oxidative metabolism of the endocannabinoid anandamide. Based on the presence of enzymes that conjugate fatty acids with glycine and the high abundance of palmitic acid in the brain, we hypothesized the endogenous formation of the saturated N-acyl amide N-palmitoyl glycine (PalGly). PalGly was partially purified from rat lipid extracts and identified using nano-high-performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry. Here, we show that PalGly is produced after cellular stimulation and that it occurs in high levels in rat skin and spinal cord. PalGly was up-regulated in fatty acid amide hydrolase knockout mice, suggesting a pathway for enzymatic regulation. PalGly potently inhibited heat-evoked firing of nociceptive neurons in rat dorsal horn. In addition, PalGly induced transient calcium influx in native adult dorsal root ganglion (DRG) cells and a DRG-like cell line (F-11). The effect of PalGly on the latter cells was characterized by strict structural requirements, PTX sensitivity, and dependence on the presence of extracellular calcium. PalGly-induced calcium influx was blocked by the nonselective calcium channel blockers ruthenium red, 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SK&F96365), and La3+. Furthermore, PalGly contributed to the production of NO through calcium-sensitive nitric-oxide synthase enzymes present in F-11 cells and was inhibited by the nitric-oxide synthase inhibitor 7-nitroindazole.

Effect of Flunarizine, a Calcium Channel Blocker, on Intraocular Pressure and Aqueous Humor Dynamics in Monkeys

To evaluate the effects of flunarizine, a nonselective calcium channel blocker, on intraocular pressure (IOP) in monkeys with laser-induced unilateral glaucoma and on aqueous humor dynamics in normal monkeys. The IOP was measured before and hourly for 6 hours after single-dose administration of 0.5%, 1%, or 2% flunarizine to the glaucomatous eye of 8 monkeys with unilateral laser-induced glaucoma. In a separate multiple-dose study, 0.5% flunarizine was applied twice daily for 5 consecutive days to the glaucomatous eye of the same 8 monkeys. IOP was measured at untreated baseline, after treatment with vehicle only, and on treatment days 1, 3, and 5. Tonographic outflow facility and fluorophotometric flow rates of aqueous humor were measured in 7 normal monkeys before and after the fifth dose of twice-daily treatment with 0.5% flunarizine. Unilateral application of 50 microL of 0.5%, 1%, or 2% flunarizine reduced IOP bilaterally. In the treated glaucomatous eyes, flunarizine reduced the IOP for 2, 3, or 5 hours, with a maximum reduction of 2.5+/-0.5 (mean+/-SEM) mm Hg (9%), 3.0+/-0.4 mm Hg (10%), and 5.0+/-0.8 mm Hg (18%) following the 0.5%, 1%, and 2% concentrations, respectively (P<0.01). The maximum reductions in IOP in the contralateral untreated eyes were 1.3+/-0.5 mm Hg, 1.5+/-0.3 mm Hg, and 2.9+/-0.7 mm Hg following the 0.5%, 1%, and 2% concentrations, respectively (P<0.05). Both the magnitude and duration of the ocular hypotensive effect of 0.5% flunarizine were enhanced with twice-daily administration for 5 days. Outflow facility in normal monkey eyes was increased (P<0.05) by 39% in the treated eyes compared with vehicle-treated contralateral eyes and by 41% compared with baseline values, and aqueous humor flow rates were unchanged (P>0.30). Flunarizine reduces IOP in a dose-dependent manner when administered to glaucomatous monkey eyes, but also has an ocular hypotensive effect on the contralateral untreated eyes. An increase in tonographic outflow facility seems to account for the IOP reduction in normal monkey eyes.

Involvement of multiple signaling pathways in PACAP-induced EM66 secretion from chromaffin cells

Secretoneurin (SN) and EM66 are two highly conserved peptides that derive from the processing of secretogranin II (SgII), one of the major constituents of chromaffin cell secretory vesicles. It has been shown that PACAP regulates SgII gene transcription and SN release in bovine adrenochromaffin cells. The aim of the present study was to localize and characterize EM66 in the bovine adrenal gland, and to examine the signaling pathways activated by PACAP to regulate the secretion of EM66 from cultured chromaffin cells. Double immunohistochemical labeling showed an intense EM66-immunoreactive (EM66-IR) signal in TH-positive medullary chromaffin cells of the adrenal gland. HPLC analysis combined with RIA detection revealed, in adrenal medulla extracts and cultured chromaffin cell media, the presence of a major EM66-IR peak co-eluting with the recombinant peptide. PACAP dose-dependently stimulated EM66 release from chromaffin cells (ED 50 = 4.8 nM). The effect of PACAP on EM66 secretion was observed after 6 h of treatment and increased to reach a 2.6-fold stimulation at 48 h. The nonselective calcium channel blocker NiCl 2, the cytosolic calcium chelator BAPTA-AM and the L-type calcium channel blocker nimodipine significantly inhibited the stimulatory effect of PACAP on EM66 release. The secretory response to PACAP was also significantly lowered by the protein kinase A inhibitor H89 and by the protein kinase C inhibitor chelerythrine. Concomitant administration of chelerythrine, H89, NiCl 2 and BAPTA totally abolished PACAP-stimulated EM66 secretion. The MAPK inhibitors U0126 and SB203580 respectively decreased by 63% and 72% PACAP-evoked EM66 release. These results indicate that, in bovine adrenal medulla, SgII is processed to generate the EM66 peptide and that PACAP activates multiple signaling pathways to regulate EM66 release from chromaffin cells, suggesting that EM66 may act downstream of the trans-synaptic stimulation of the adrenal medulla by neurocrine factors.

Attenuation by 4-aminopyridine of delayed vasorelaxation by troglitazone

Troglitazone and other thiazolidinediones (TZDs) are thought to relax arterial smooth muscle by directly inhibiting calcium channels in smooth muscle cell membranes. However, until recently such inhibition was only examined acutely, ie, within only seconds or minutes after administration of these agents to arterial smooth muscle preparations. Recently, a novel experiment was reported in which troglitazone caused a 2-phase relaxation of perfused resistance arteries, namely, an acute relaxation (within the first 20 minutes of treatment), which was blocked by a nonselective calcium channel blocker and a delayed relaxation (after 2 hours), which was not. We sought to determine if any of the 4 major potassium (K) channels in vascular smooth muscle play a role in the delayed relaxation. We incubated vascular contractile rings prepared from ventral tail arteries of rats with physiological buffer containing either 0 or 4 μmol/L troglitazone for 3 hours (4 μmol/L is typical of plasma levels from diabetic patients). Different K channel inhibitors (1 mmol/L 4-aminopyridine [4AP]; 1 mmol/L tetraethylammonium [TEA]; 5 μmol/L glyburide; 20 μmol/L barium) were coadministered with each level of troglitazone in additional preparations. Then these arterial rings were contracted with either norepinephrine (NE), arginine vasopressin (AVP), or high-K buffer. All contractions were significantly relaxed by troglitazone ( P < .05). Only 4AP significantly attenuated troglitazone’s relaxation of NE and AVP contractions ( P < .05), though not high-K-induced contractions. TEA, glyburide, and barium had no such influence. Thus, for both adrenergic (NE) and nonadrenergic (AVP) contractions, the delayed arterial vasorelaxation by troglitazone may be mediated at least in part by activation of 4AP-sensitive K channels. Furthermore, the specific subtype of the channels involved is most likely those bound in the outer cell membrane where their effectiveness in terms of mediating relaxation would depend on an intact transmembrane K ion gradient.

Analysis of non-selective calcium-channel blockers by reversed-phase TLC

The selectivity of TLC separation of non-selective calcium-channel blockers prenylamine, lidoflazine, bepridil, and fendiline has been investigated silanized silica gel RP8 and RP18 plates. Optimization of retention and selectivity for these compounds was achieved by altering the pH and the concentration of organic modifier (methanol, ethanol, tetrahydrofuran, acetonitrile) in the aqueous mobile phases. The substances were separated in horizontal chambers and the drugs were detected by videoscanning and illumination of the plates at λ = 254 nm. On RP8 plates the best separation was achieved with 50% acetonitrile in pH 2.06 phosphate buffer as mobile phase. On RP18 the best separation was achieved with 50% ethanol in pH 2.06 phosphate buffer.

Analysis of some nonselective calcium-channel blockers in normal-phase chromatographic systems

The nonselective calcium-channel blockers prenylamine, lidoflazine, bepridil, and fendiline have been separated by TLC on silica gel 60F 254 , alumina 60F 254 and Florisil with several mobile phases comprising mixtures of n -hexane with polar modifiers such as 2,4-dioxane, ethyl acetate, methanol, ethyl methyl ketone, tetrahydrofuran, and propan-2-ol. Plates were developed in horizontal chambers and visualized under UV irradiation at λ = 254 nm, by use a videoscanning system, and by use of different reagents. The best separation was achieved on alumina 60F 254 plates with tetrahy-drofuran-hexane, 2 + 8 ( v / v ) as mobile phase.

Effect of calcium on the vascular contraction induced by cobra venom cardiotoxin.

1. The cytotoxic effects of cardiotoxin (CTX) purified from Cobra venom were tested in endothelium-denuded rat aortic ring preparations in tissue organ baths and the effect of extracellular Ca2+ on the cytotoxic effect of CTX was investigated using a digital dynamic calcium imaging technique. 2. At 10 micromol/L, CTX induced a slowly developing and sustained contraction that amounted to approximately 50% of the maximal contraction induced by 80 mmol/L KCl. At high concentrations (> 15 micromol/L), CTX caused irreversible damage to the smooth muscle contractile function. However, washout of CTX at its peak contraction did not affect the subsequent contraction to either KCl or phenylephrine. 3. Contraction induced by CTX was dependent on the Ca2+ concentration in the external solution. A maximal contractile response to CTX was obtained in medium containing 1-2.5 mmol/L Ca2+. This contractile response induced by CTX decreased with higher Ca2+ concentrations and was completely diminished when 7 mmol/L Ca2+, 3 mmol/L Ni2+ or 30 micromol/L tetrandrine (a non-selective calcium channel blocker) was present in the external solution before addition of CTX to the bath. 4. The above observations were supported by the calcium imaging work performed with cultured aortic smooth muscle cells from Wistar-Kyoto rats, in which CTX was shown to induce the elevation of cytosolic Ca2+ in the presence, but not in the absence, of 2.5 mmol/L extracellular Ca2+. Increasing the extracellular Ca2+ concentration to 7 mmol/L, the addition of 3 mmol/L Ni2+ or inclusion of 30 micro mol/L tetrandrine inhibited the elevation of cytosolic Ca2+ induced by CTX. 5. These results suggest that: (i) a CTX-sensitive internal calcium store does not exist in rat aortic smooth muscle; (ii) the contractile effect CTX is associated with a Ca2+ influx process; and (iii) CTX interacts extracellularly with the plasma membrane at the level of the calcium channels, as well as anionic sites to which Ca2+ and other inorganic cations bind.

Calcium Waves Induced by Large Voltage Pulses in Fish Keratocytes

Intracellular calcium waves in fish keratocytes are induced by the application of electric field pulses with amplitudes between 55 and 120 V/cm and full width at half-maximum of 65–100 ms. Calcium concentrations were imaged using two-photon excited fluorescence microscopy (Denk et al., 1990. Science. 248:73–76; Williams et al., 1994. FASEB J. 8:804–813) and the ratiometric calcium indicator indo-1. The applied electric field pulses induced waves with fast calcium rise times and slow decays, which nucleated in the lamellipodium at the hyperpolarized side of the cells and, less frequently, at the depolarized side. The effectiveness of wave generation was determined by the change induced in the membrane potential, which is about half the field strength times the cell width in the direction of the field. Stimulation of waves began at voltage drops across the cell above 150 mV and saturated at voltage drops above 300 mV, where almost all cells exhibited a wave. Waves were not induced in low-calcium media and were blocked by the nonselective calcium channel blockers cobalt chloride and verapamil, but not by specific organic antagonists of voltage-sensitive calcium channel conductance. Thapsigargin stopped wave propagation in the cell body, indicating that calcium release from intracellular stores is necessary. Thus a voltage pulse stimulates Ca 2+ influx through calcium channels in the plasma membrane, and if the intracellular calcium concentration reaches a threshold, release from intracellular stores is induced, creating a propagating wave. These observations and the measured parameters (average velocity ∼66 μm/s and average rise time ∼68 ms) are consistent with a wave amplification model in which {formula not available us MathML} determines the effective diffusivity of the propagating molecules, D ≈ 300 μm 2/s (Meyer, 1991. Cell. 64:675–678).

Inhibition of Na+,K+‐ATPase by Ouabain Opens Calcium Channels Coupled to Acetylcholine Release in Guinea Pig Myenteric Plexus

Ouabain, an Na+K+ATPase inhibitor, increases the release of acetylcholine (ACh) from various preparations in a Ca2+ -independent way. However, in other preparations the release of ACh evoked by ouabain is dependent on the presence of extracellular calcium. In the present study, we have labeled the ACh of myenteric plexus longitudinal muscles of guinea pig ileum and compared the effect of calcium channel blockers on ouabain-evoked release of [3H]ACh. Release of [3H]ACh evoked by ouabain is dose dependent and decreased markedly in the absence of calcium or in the presence of cadmium, a nonspecific calcium channel blocker. N-type calcium channel blockage by the omega-conotoxins GVIA (selective N-type calcium channel blocker) and MVIC (a nonselective calcium channel blocker) inhibited by 45 and 55%, respectively, the release of [3H]ACh. L-type calcium channel suppression by low concentrations of verapamil, nifedipine, and diltiazem had no effect on the release of [3H]ACh. The release of transmitter was also not affected significantly by nickel, a T-type calcium channel blocker. In addition, omega-agatoxin-IVA, at concentrations that block P- and Q-type calcium channels, did not affect significantly the release of [3H]ACh. Thus, extracellular Ca2+ is essential for the release of ACh induced by ouabain from guinea pig ileum myenteric plexus. In this preparation, the N-type calcium channel plays a dominant role in transmitter release evoked by inhibition of Na+K+-ATPase, but other routes of calcium entry in addition to these channels can also support the release of neurotransmitter induced by ouabain.

Endothelin-induced activation of neutrophil migration

Migration of rabbit peritoneal neutrophils was stimulated by endothelin-1 (ET-1) up to 2.10 −8M. Higher concentrations inhibited random migration. The stimulating effect of ET-1 was inhibited by BQ-123, a specific antagonist of the ET A receptor. A checkerboard assay showed that the stimulating effect of ET-1 on neutrophil migration was chemokinetic rather than chemotactic. Extracellular Ca 2+ was required for the activating effect of ET-1. Non-selective calcium channel blockers such as econazole and La 3+ strongly inhibited ET-1-activated migration but had little effect on fMLP-activated migration, underlining the importance of Ca 2+ influx for ET-1-activated migration. Studies with electroporated neutrophils showed that the increase in migration was most pronounced at calcium concentrations between 100 nM and 1 μM. ET-1-activated migration of electroporated cells was completely blocked by low concentrations of calcium-channel blockers such as verapamil and nitrendipine. Migration by intact cells was inhibited by the same concentration of verapamil, but to a lesser degree; nitrendipine had little effect on migration of intact cells. This suggests that calcium derived from intracellular stores is required for migration activated by ET-1. Protein kinase C, protein tyrosine kinase, and phosphatase activity were involved in the activating effect of ET-1 on neutrophil migration. ET-1 did not induce exocytotic enzyme release, in neither the presence nor the absence of cytochalasin B.

ω-Conotoxin- and nifedipine-insensitive voltage-operated calcium channels mediate K +-induced release of pro-thyrotropin-releasing hormone-connecting peptides Ps4 and Ps5 from perifused rat hypothalamic slices

The rat thyrotropin-releasing hormone (TRH) precursor (prepro-TRH) contains five copies of the TRH progenitor sequence linked together by intervening sequences. Recently, we have shown that the connecting peptides prepro-TRH-(160–169) (Ps4) and prepro-TRH-(178–199) (Ps5) are released from rat hypothalamic neurones in response to elevated potassium concentrations, in a calcium-dependent manner. In the present study, the role of voltage-operated calcium channels in potassium-induced release of Ps4 and Ps5 was investigated, using a perifusion system for rat hypothalamic slices. The release of Ps4 and Ps5 stimulated by potassium (70 mM) was blocked by the inorganic ions Co 2+ (2.6 mM) and Ni 2+ (5 mM). In contrast, the stimulatory effect of KCl was insensitive to Cd 2+ (100 μM). The dihydropyridine antagonist nifedipine (10 μM) had no effect on K +-evoked release of Ps4 and Ps5. Furthermore, the response to KCl was not affected by nifedipine (10 μM) in combination with diltiazem (1 μM), a benzothiazepine which increases the affinity of dihydropyridine antagonists for their receptor. The dihydropyridine agonist BAY K 8644, at concentrations as high as 1 mM, did not stimulate the basal secretion of Ps4 and Ps5. In addition, BAY K 8644 had no potentiating effect on K +-induced release of Ps4 and Ps5. The marine cone snail toxin ω-conotoxin, a blocker of both L- and N-type calcium channels had no effect on the release of Ps4 and Ps5 stimulated by potassium. Similarly, the ω-conopeptide SNX-111, a selective blocker of N-type calcium channels, did not inhibit the stimulatory effect of potassium. The release of Ps4 and Ps5 evoked by high K + was insensitive to the non-selective calcium channel blocker verapamil (20 μM). Amiloride (1 μM), a putative blocker of T-type calcium channels, did not affect KCl-induced secretion of the two connecting peptides. Taken together, these results indicate that two connecting peptides derived from the pro-TRH, Ps4 and Ps5, are released by K +-induced depolarization through activation of voltage-sensitive calcium channels. The calcium channels appear to have a pharmacological profile different from that of L- and N-type channels. Although, their insensitivity to low Cd 2+ concentrations and sensitivity to Ni 2+ ions would support the involvement of T-type calcium channels, the lack of effect of amiloride suggests that they belong to a yet undefined class of calcium channels.

Renal hemodynamic and natriuretic effects of manganese and interactions with atrial natriuretic peptide

Manganese (Mn2+) is a cofactor for guanylate cyclase (GC), which is involved in the generation of guanosine 3',5'-cyclic monophosphate (cGMP), a second messenger for atrial natriuretic peptide (ANP) action. Mn2+ is also, however, a nonselective calcium-channel blocker. We examined the effects of infusion of MnCl2 into normal rats and its interaction in vivo and in vitro with GC and ANP. MnCl2 significantly increased glomerular filtration rate (GFR) and effective renal plasma flow rate (RPF). These effects were caused by selective afferent arteriolar vasodilatation, which allowed the glomerular capillary plasma flow rate and hydraulic pressure to rise, thus elevating single-nephron GFR. Urinary Na+ excretion (UNaV) also increased with MnCl2. The natriuresis was, unlike ANP, not mediated by GC activation and cGMP production, as MnCl2 had no effect on either urinary cGMP excretion or cGMP accumulation in intact inner medullary collecting duct cell (IMCD) suspensions, nor did it affect Na(+)-dependent oxygen consumption in these cells. When superimposed on an infusion of ANP, MnCl2 resulted in significant increases in UNaV, GFR, and RPF. These effects were associated with small but significant increments in urinary cGMP excretion. However, MnCl2 did not affect in vitro cGMP production in intact IMCDs or glomeruli in response to ANP stimulation. It is uncertain therefore whether the in vivo augmentation of the natriuretic effect of ANP by MnCl2 is related to GC activation and cGMP production.

Voltage-regulated calcium channels involved in the regulation of enkephalin synthesis are blocked by phorbol ester treatment.

Treatment of bovine chromaffin cells with 40 mM KCl stimulates a 3-fold increase in total methionine enkephalin immunoreactivity (medium plus cells) and a 4-fold increase in proenkephalin mRNA (mRNAenk). These effects of KCl, which are dependent on extracellular calcium, can be blocked by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), although release of methionine enkephalin appears less affected. Using fura-2-loaded chromaffin cells and a dual-excitation wavelength spectrofluorometer, we have examined whether the actions of KCl and TPA on methionine enkephalin synthesis and release can be explained by changes in intracellular free calcium ([Ca2+]i). KCl produced a rapid 600 nM increase in [Ca2+]i from resting levels of approximately 170 nM. Subsequently, [Ca2+]i declined to a new steady-state plateau which was approximately 275 nM higher than the original resting levels. The postdepolarization plateau of [Ca2+]i was reduced by TPA, (-)-(R)-202,791 (a dihydropyridine calcium channel antagonist), and LaCl3 (a nonselective calcium channel blocker). TPA also inhibited potentiation of the KCl-stimulated plateau of [Ca2+]i due to (+)-(S)-202,791, a calcium channel agonist. In contrast, TPA had no effect on resting [Ca2+]i and only slightly inhibited the initial rapid KCl-stimulated increase in [Ca2+]i. The inhibitory effects were maintained for 24 h in the continuous presence of TPA. We conclude 1) that TPA inhibits enkephalin synthesis by inactivating dihydropyridine-sensitive voltage-dependent calcium channels, 2) that these channels alone maintain elevated [Ca2+]i following KCl depolarization, and 3) that sustained elevation in [Ca2+]i is necessary in order to increase enkephalin synthesis in KCl-treated chromaffin cells.