OBJECTIVETo determine the expression levels of calcium channel alpha-2-delta-1 (Cavalpha2delta-1) subunit in trigeminal ganglia (TG) and trigeminal spinal subnucleus caudalis C1-C2 spinal cervical dorsal horn(Vc/C2) after infraorbital nerve (ION) ligation injury in relation to the development of neuropathic nociception.

METHODSTwelve male SD rats were divided into two groups randomly, with 6 rats for each. In the ligation group, the ION was ligated by chromic gut loosely. In the sham operation group, the ION was exposed under same procedure but not ligated. The behavioral tests were conducted before 1 d and after 3, 6, 9, 12, 15 d with calibrated von Frey filaments. Cavalpha2delta-1 protein levels in TG and Vc/C2 were measured with Western blots 15 days after surgery.

RESULTSCompared with the sham operation group, changes of allodynia to mechanical stimulation on the territory of ligated ION were found from the 9th to 15th day after operation in the ligation group (P<0.05). The expression levels of Cavalpha2delta-1 in TG and Vc/ C2 after ION ligation injury upregulated significantly (P<0.01).

CONCLUSIONThe expression levels of Cavalpha2delta-1 in TG and Vc/C2 after ION ligation injury were upregulated and it may contribute to the pathogenesis of neuropathic nociception.

Animals ; Calcium ; Calcium Channels ; Calcium Channels, L-Type ; Male ; Rats ; Rats, Sprague-Dawley ; Trigeminal Neuralgia

Voltage gated calcium channels (VGCCs) are multi-subunit membrane proteins present in a variety of tissues and control many essential physiological processes. Due to their vital importance, VGCCs are regulated by a myriad of proteins and signaling pathways. Here we review the literature on the regulation of VGCCs by proteolysis of the pore-forming α1 subunit, Ca(v)α(1). This form of regulation modulates channel function and degradation and affects cellular gene expression and excitability. L-type Ca(2+) channels are proteolyzed in two ways, depending on tissue localization. In the heart and skeletal muscle, the distal C-terminus of Ca(v)α(1) is cleaved and acts as an autoinhibitor when it reassociates with the proximal C-terminus. Relief of this autoinhibition underlies the β-adrenergic stimulation-induced enhancement of cardiac and skeletal muscle calcium currents, part of the "fight or flight" response. Proteolysis of the distal C-terminus of L-type channels also occurs in the brain and is probably catalyzed by a calpain-like protease. In some brain regions, the entire C-terminus of L-type Ca(2+) channels can be cleaved by an unknown protease and translocates to the nucleus acting as a transcription factor. The distal C-terminus of P/Q-channel Ca(v)α(1) is also proteolyzed and translocates to the nucleus. Truncated forms of the PQ-channel Ca(v)α(1) are produced by many disease-causing mutations and interfere with the function of full-length channels. Truncated forms of N-type channel Ca(v)α(1), generated by mutagenesis, affect the expression of full-length channels. New forms of proteolysis of VGCC subunits remain to be discovered and may represent a fruitful area of VGCC research.
Animals ; Calcium Channels, L-Type ; metabolism ; Calcium Signaling ; Humans ; Muscle, Skeletal ; physiology ; Proteolysis

Voltage dependent calcium channel (VDCC), one of the most important regulator of Ca2+ concentration in neuron, play an essential role in the central processing of nociceptive information. The present study investigated the antinociceptive effects of L, T or N type VDCC blockers on the formalin-induced orofacial inflammatory pain. Experiments were carried out on adult male Sprague-Dawley rats weighing 220-280 g. Anesthetized rats were individually fixed on a stereotaxic frame and a polyethylene (PE) tube was implanted for intracisternal injection. After 72 hours, 5% formalin (50 microL) was applied subcutaneously to the vibrissa pad and nociceptive scratching behavior was recorded for nine successive 5 min intervals. VDCC blockers were administered intracisternally 20 minutes prior to subcutaneous injection of formalin into the orofacial area. The intracisternal administration of 350 or 700 microg of verapamil, a blocker of L type VDCC, significantly decreased the number of scratches and duration in the behavioral responses produced by formalin injection. Intracisternal administration of 75 or 150 microg of mibefradil, a T type VDCC blocker, or 11 or 22 microg of cilnidipine, a N type VDCC blocker, also produced significant suppression of the number of scratches and duration of scratching in the first and second phase. Neither intracisternal administration of all VDCC blockers nor vehicle did not affect in motor dysfunction. The present results suggest that central VDCCs play an important role in orofacial nociceptive transmission and a targeted inhibition of the VDCCs is a potentially important treatment approach for inflammatory pain originating in the orofacial area.
Adult ; Animals ; Calcium ; Calcium Channel Blockers ; Calcium Channels ; Calcium Channels, L-Type ; Calcium Channels, N-Type ; Calcium Channels, T-Type ; Dihydropyridines ; Facial Pain ; Formaldehyde ; Humans ; Injections, Subcutaneous ; Male ; Mibefradil ; Neurons ; Pain Measurement ; Polyethylene ; Rats ; Rats, Sprague-Dawley ; Verapamil

OBJECTIVETo explore the mechanisms of the influx of calcium ions during the activation of ACh-sensitive BK channel (big conductance, calcium-dependent potassium channel) in type II vestibular hair cells of guinea pigs.

METHODSType II vestibular hair cells were isolated by collagenase type IA. Under the whole-cell patch mode, the sensitivity of ACh-sensitive BK current to the calcium channels blockers was investigated, the pharmacological property of L-type calcium channel activator-sensitive current and ACh-sensitive BK current was compared.

RESULTSFollowing application of ACh, type II vestibular hair cells displayed a sustained outward potassium current, with a reversal potential of (-70.5 +/- 10.6) mV (x +/- s, n = 10). At the holding potential of -50 mV, the current amplitude of ACh-sensitive potassium current activated by 100 micromol/L ACh was (267 +/- 106) pA (n = 11). ACh-sensitive potassium current was potently sensitive to the BK current blocker, IBTX (iberiotoxin, 200 nmol/L). Apamin, the well-known small conductance, calcium-dependent potassium current blocker, failed to inhibit the amplitude of ACh-sensitive potassium current at a dose of 1 micromol/L. ACh-sensitive BK current was sensitive to NiCl2 and potently inhibited by CdCl2. NiCl2 and CdCl2 showed a dose-dependent blocking effect with a half inhibition-maximal response of (135.5 +/- 18.5) micromol/L (n = 7) and (23.4 +/- 2.6) micromol/L (n = 7). The L-type calcium channel activator, (-) -Bay-K 8644 (10 micromol /L), mimicked the role of ACh and activated the IBTX-sensitive outward current.

CONCLUSIONACh-sensitive BK and L-type calcium channels are co-located in type II vestibular hair cells of guinea pigs.

Animals ; Calcium Channels, L-Type ; Guinea Pigs ; Hair Cells, Vestibular ; metabolism ; Large-Conductance Calcium-Activated Potassium Channels ; Patch-Clamp Techniques

Melittin-induced nociceptive responses are mediated by selective activation of capsaicin-sensitive primary afferent fibers and are modulated by excitatory amino acid receptor, cyclooxygenase, protein kinase C and serotonin receptor. The present study was undertaken to investigate the peripheral and spinal actions of voltage-gated calcium channel antagonists on melittin-induced nociceptive responses. Changes in mechanical threshold and number of flinchings were measured after intraplantar (i.pl.) injection of melittin (30microg/paw) into mid-plantar area of hindpaw. L-type calcium channel antagonists, verapamil [intrathecal (i.t.), 6 or 12microg; i.pl.,100 & 200microg; i.p., 10 or 30 mg], N-type calcium channel blocker, omega-conotoxin GVIA (i.t., 0.1 or 0.5microg; i.pl., 5microg) and P-type calcium channel antagonist, omega-agatoxin IVA (i.t., 0.5microg; i.pl., 5microg) were administered 20 min before or 60 min after i.pl. injection of melittin. Intraplantar pre-treatment and i.t. pre- or post-treatment of verapamil and omega-conotoxin GVIA dose-dependently attenuated the reduction of mechanical threshold, and melittin-induced flinchings were inhibited by i.pl. or i.t. pre-treatment of both antagonists. P-type calcium channel blocker, omega-agatoxin IVA, had significant inhibitory action on flinching behaviors, but had a limited effect on melittin-induced decrease in mechanical threshold. These experimental findings suggest that verapamil and omega-conotoxin GVIA can inhibit the development and maintenance of melittin-induced nociceptive responses.
Animals ; Calcium Channels* ; Calcium Channels, L-Type ; Calcium Channels, N-Type ; Calcium Channels, P-Type ; Calcium* ; Hyperalgesia ; Ions* ; Melitten ; Nociception* ; omega-Agatoxin IVA ; omega-Conotoxin GVIA ; Prostaglandin-Endoperoxide Synthases ; Protein Kinase C ; Rats* ; Receptors, Glutamate ; Serotonin ; Verapamil

PURPOSE: This study was designed to investigate the effects of polyamines on rabbit seminal vesicular contractility. MATERIALS AND METHODS: The polyamines; putrescine, spermidine and spermine, were added to deepithelized and precontracted seminal vesicle strips, with either 10 4M norepinephrine (NE), 10 4M acetylcholine (ACh) or 70mM KCl, in organ chambers to obtain cumulative concentration response curves. A whole cell mode patch clamp study was also performed to observe the effects of the polyamines on the L-type calcium channel activities. RESULTS: The polyamines elicited concentration-dependent relaxations of the precontracted strips with the NE, ACh and KCl. The spermine showed the most potent relaxation response. Both extracellular and intracellular application of the spermine decreased the L-type calcium channel currents. CONCLUSIONS: Spermine more potently inhibited the seminal vesicle contraction than putrescine or spermidine, which suggests the polyamines may play a role in maintaining the basal tonicity of seminal vesicle in a flaccid state. The spermine-induced relaxation response seems to be related with an inhibition of the L-type calcium channel activities.
Acetylcholine ; Calcium Channels, L-Type ; Norepinephrine ; Polyamines* ; Putrescine ; Relaxation* ; Seminal Vesicles* ; Spermidine ; Spermine

BACKGROUND: Ketamine causes a dose-dependent vasodilation of a norepinephrine-precontracted pulmonary arterial ring. The goals of the present in vitro study were to investigate the effects of ketamine on phenylephrine-induced contraction and to elucidate its cellular mechanism. METHODS: Following endothelial denudation, isolated thoracic aortic rings were suspended for isometric tension recording. Contractile response to 60 mM KCl (potassium chloride) was measured in the absence or presence of ketamine (5x10(-5), 10(-4), 10(-3) M) in the rings. Phenylephrine dose (10(-8) to 10(-5) M)-response curves were generated in the absence or presence of ketamine. In rings pretreated with verapamil (10(-5) M) or with combined verapamil (10(-5) M) and ryanodine (2x10(-5) M), phenylephrine dose-response curves were also generated in the absence or presence of ketamine. RESULTS: Ketamine attenuated the contractile response to 60 mM KCl compared with rings without ketamine in a concentration-dependent manner, and ketamine (5x10(-5), 10(-4), 10(-3)M) caused a rightward shift of the dose-response curve to phenylephrine in a concentration-dependent manner. In verapamil 10(-5) M pretreated rings, ketamine 10(-3) M attenuated phenylephrine-induced contraction compared to rings without ketamine, but a low-dose of ketamine (5x10(-5), 10(-4)M) had no effect. In rings pretreated with combined verapamil 10(-5) M and ryanodine 2 x 10(-5) M, ketamine 10(-3) M attenuated phenylephrine-induced contraction compared with rings without ketamine. CONCLUSIONS: These results indicate that clinically relevant concentration (5 x 10(-5) M) of ketamine attenuates phenylephrine-induced contraction by inhibiting the L-type calcium channel.
Animals ; Aorta, Thoracic* ; Calcium Channels, L-Type ; Ketamine* ; Phenylephrine ; Rats* ; Ryanodine ; Vasodilation ; Verapamil

This study is aimed to investigate the effects of high intracellular Mg²⁺ on L-type calcium channel in guinea-pig ventricular myocytes. The cardiomyocytes were acutely isolated with enzyme digestion method. By adopting inside-out configuration of patch clamp technique, single channel currents of the L-type calcium channel were recorded under different intracellular Mg²⁺ concentrations ([Mg²⁺]i). In control group, which was treated with 0.9 mmol/L Mg²⁺, the relative activity of calcium channel was (176.5 ± 34.1)% (n = 7). When [Mg²⁺]i was increased from 0.9 to 8.1 mmol/L (high Mg²⁺ group), the relative activities of calcium channel decreased to (64.8 ± 18.1)% (n = 6, P < 0.05). Moreover, under 8.1 mmol/L Mg²⁺, the mean open time of calcium channel was shortened to about 25% of that under control condition (P < 0.05), but the mean close time of calcium channel was not altered. These results suggest that high intracellular Mg²⁺ may inhibit the activities of L-type calcium channel, which is mainly due to the shortening of the mean open time of single L-type calcium channel.
Animals ; Calcium Channels, L-Type ; physiology ; Guinea Pigs ; Magnesium ; physiology ; Myocytes, Cardiac ; physiology ; Patch-Clamp Techniques

BACKGROUND: It has been shown that L-type calcium channel blockers increase the muscle relaxation effects of non-depolarizing neuromuscular blocking agents whereas the potentiated neuromuscular blocking effects by L-type calcium channel blocker are resistant to reversal by neostigmine. The aims of this study were 1) to see whether the pretreatment of L-type calcium channel blocker, such as verapamil, aggravates the pipecuronium-induced muscle relaxation, 2) if so, to see whether these effects are reversed by anticholinesterase, such as neostigmine and edrophonium or potassium channel blocker, such as 4-aminopyridine. METHODS: The rat-phrenic nerve-hemidiaphragms (n=60) were prepared. Twenty microgram of pipecuronium was administered to all organ bath. All samples were divided into two groups according to the administration of 10uM of verapamil i.e. verapamil pretreated, non-pretreated group. The amounts of administered pipecuronium were gradually increased by 4ug until the force of twitch decreased to 10% of control value in both groups. Each group was subdivided into three groups according to the administration of 0.75 M of neostigmine, 12.4 uM of edrophonium or 40uM of 4-aminopyridine. RESULTS: The dose of pipecuronium required for the decrease of contractile force to 10% of control value was less in verapamil pretreated group than in non-pretreated group. And, the decrease of contractile force in both groups was more effectively reversed by 4-aminopyridine than neostigmine and edrophonium. CONCLUSIONS: Verapamil potentiates the pipecuronium-induced neuromuscular blockade and 4-aminopyridine is more effective to reverse verapamil pretreated, pipecuronium induced neuromuscular blockade.
4-Aminopyridine* ; Baths ; Calcium Channels, L-Type ; Edrophonium* ; Muscle Relaxation ; Neostigmine* ; Neuromuscular Blockade* ; Neuromuscular Blocking Agents ; Pipecuronium* ; Potassium Channels ; Verapamil*

BACKGROUNDThe contractility of hepatic stellate cells (HSCs) may play an important role in the pathogenesis of cirrhosis with portal hypertension. The aim of this study was to research the effects of octreotide, an analogue of somatostatin, on intracellular Ca2+ and on the expression of L-type voltage-operated calcium channels (L-VOCCs) in activated HSCs, and to try to survey the use of octreotide in treatment and prevention of cirrhosis with portal hypertension complications.

METHODSHSC-T6, an activated HSCs line, was plated on small glass coverslips in 35-mm culture dishes at a density of 1 x 10(5)/ml, and incubated in DMEM media for 24 hours. After the cells were loaded with Fluo-3/AM, intracellular Ca2+ was measured by Laser Scanning Confocal Microscopy (LSCM). The dynamic changes in activated HSCs of intracellular Ca2+, stimulated by octreotide, endothelin-1, and KCl, respectively, were also determined by LSCM. Each experiment was repeated six times. L-VOCC expression in HSCs was estimated by immunocytochemistry.

RESULTSAfter octreotide stimulation, a significant decrease in the intracellular Ca2+ of activated HSCs was observed. However, octreotide did not inhibit the increases in intracellular Ca2+ after stimulation by KCl and endothelin-1. Moreover, octreotide did not significantly affect L-VOCC expression. These results suggest that neither L-VOCC nor endothelin-1 receptors in activated HSCs are inhibited by octreotide.

CONCLUSIONSOctreotide may decrease portal hypertension and intrahepatic vascular tension by inhibiting activated HSCs contractility through decreases in intracellular Ca2+. The somatostatin receptors in activated HSCs may be inhibited by octreotide.

Calcium ; analysis ; Calcium Channels, L-Type ; analysis ; Cells, Cultured ; Hepatocytes ; chemistry ; cytology ; drug effects ; Microscopy, Confocal ; Octreotide ; pharmacology