Dihydropyridine sensitive calcium channels in a smooth muscle cell line

The pharmacological properties of voltage sensitive calcium channels (VSCC) were examined in a rat aortic smooth muscle cell line (A10). The inorganic VSCC blockers Co2+ and Cd2+ blocked 45Ca2+ uptake into these cells in both 5 mM K+ and 50 mM K+ (depolarizing) conditions. The organic VSCC antagonists nitrendipine, nimodipine, D-600 and diltiazem also blocked 45Ca2+ uptake at low concentrations. The relative potencies of blockade were similar to those found in intact vascular smooth muscle. The VSCC "agonist" BAY K8644 enhanced 45Ca2+ uptake and this effect could be reversed by nitrendipine. These results indicate that A10 cells possess VSCC and that these VSCC behave similarly to those in authentic smooth muscle.     

Mitochondrial genotype in vulvar carcinoma - cuckoo in the nest

Vulvar squamous cell carcinoma (VSCC) is a rare female genital neoplasm. Although numerous molecular changes have been reported in VSCC, biomarkers of clinical relevance are still lacking. On the other hand, there is emerging evidence on the use of mtDNA as a diagnostic tool in oncology. In order to investigate mtDNA status in VSCC patients, haplogroup distribution analysis and D-loop sequencing were performed. The results were compared with available data for the general Polish population, cancer free-centenarians as well as patients with endometrial and head and neck cancer. The obtained data were also compared with the current status of mitochondrial databases. Significant differences in haplogroup distribution between VSCC cohort, general Polish population and cancer-free centenarians cohort were found. Moreover, a correlation between the VSCC patients haplogroup and HPV status was observed. Finally, a specific pattern of mtDNA polymorphisms was found in VSCC. Our results suggest that the mitochondrial genetic background may influence the risk of VSCC occurrence as well as susceptibility to HPV infection.     

Calcium mediates dorsoventral patterning of mesoderm in Xenopus.

Calcium signals participate in the differentiation of electrically excitable and nonexcitable cells; one example of this differentiation is the acquisition of mature neuronal phenotypes. For example, transient elevations of the intracellular calcium concentration have been recorded in the ectoderm of early embryos, and this elevation has been proposed to participate in neural induction. Here, we present molecular evidence indicating that voltage-sensitive calcium channels (VSCC) are involved in early developmental processes leading to the establishment of the dorsoventral (D-V) patterning of a vertebrate embryo. We report that alpha1S VSCC are expressed selectively in the dorsal marginal zone at the early gastrula stage. The expression of the VSCC correlates with elevated intracellular calcium levels, as evaluated by the fluorescence of the intracellular calcium indicator Fluo-3. Misexpression of VSCC leads to a strong dorsalization of the ventral marginal zone and induction of the secondary axis but no direct neuralization of the ectoderm. Moreover, specific inhibition of VSCC by the use of calcicludine results in ventralization of the dorsal mesoderm. Together, these results indicate that calcium channels regulate mesodermal patterning by specificating the D-V identity of the mesodermal cells. The D-V patterning of the mesoderm has been shown to depend on a gradient of BMPs activity. We discuss the possibility that VSCC affect or act downstream of BMPs activity.     

Thyrotropin-Releasing Hormone Enhances Choline Acetyltransferase and Creatine Kinase in Cultured Spinal Ventral Horn Neurons

The effect of 0.1 mM thyrotropin-releasing hormone (TRH) on ventral horn neurons was investigated in eight experimental sets of tissue cultures established from ventral and dorsal portions of spinal cords of 13-15-day rat embryos. Cultures were treated with TRH from day 1 for 2-5 weeks. TRH-treated ventral spinal cord cultures (VSCC), compared with control VSCC, had more numerous and more healthy-appearing neurons and thicker bundles of long cell processes. In TRH-treated VSCC, choline acetyltransferase (ChAT) activity was greater than 16 times (p less than 0.005) and creatine kinase greater than 3 times (p less than 0.005) that of control VSCC. Morphologic and biochemical parameters of dorsal spinal cord cultures remained unchanged by TRH treatment. Since lower motor neurons are numerous in the ventral spinal cord (and not present in the dorsal cord) and since lower motor neurons are the major ChAT-containing spinal cord cells, our data demonstrating a beneficial effect of TRH on VSCC suggest a tropic effect of TRH on lower motor neurons.     

T-588 Enhances Neurite Outgrowth and Choline Acetyltransferase in Cultured Rat Spinal Ventral Horn Neurons

T-588(R(-)-1-(benzo(b)thiophen-5yl)-2-[2(N,N-diethylamino)ethoxy]ethanol hydrochloride) is a novel compound which has been shown to exhibit a wide range of neurotrophic effects both in vivo and in vitro. This compound can slow the motor deterioration of wobbler mouse motor neuron disease. However, it is not known whether this compound has a trophic effect on spinal motor neurons. We have studied the effect of T-588 on neurite outgrowth and choline acetyltransferase(ChAT) activity in primary explant cultures of ventral spinal cord of fetal rats(VSCC). Cultures were treated with T-588 from day 1 to 1 week. T-588 treated VSCC, compared with control VSCC, had a significant neurite promoting effect at ranged between 10^–8 molar(M) and 10^–5 M, with 2.3 to 5.3 fold increased over that of control VSCC. In T-588 treated VSCC, ChAT activity was increased 1.5 times over that of control at 10^–6, and 10^–5 M respectively. Our data showing T-588 has neurotrophic action on VSCC and suggests a potential use of T-588 in treating diseases that involve degeneration and death of spinal motor neurons, such as motor neuropathy and motor neuron disease.     

The most common chromosome aberration detected by high-resolution comparative genomic hybridization in vulvar intraepithelial neoplasia is not seen in vulvar squamous cell carcinoma

We analyzed genetic changes in condylomas (four cases), vulvar intraepithelial neoplasia I-III (VIN I-III, eleven cases), and primary vulvar squamous cell carcinomas (VSCC, ten cases) by high-resolution comparative genomic hybridization (HR-CGH) and flowcytometry. All samples were also human papilloma virus (HPV)-genotyped. Gain of chromosome 1, the aberration most often seen in VIN III (67%), was not seen in HPV-positive or -negative VSCCs (0%). Both VIN III and VSCC frequently showed gain of 3q (56 and 70%, respectively). The VIN III samples often demonstrated gain of 20q (56%) and 20p (44%), and the VSCC samples gain of 8q (60%), loss of 3p (50%), and 8p (40%). None of the four most frequent changes in the VSCC samples occurred exclusively in the HPV-positive or -negative samples. As expected, we did not find any cytogenetic changes in condylomas and nearly any changes in VIN I-II.     

Desensitization of pituitary gonadotropin secretion by agonist-induced inactivation of voltage-sensitive calcium channels

Gonadotropin-releasing hormone (GnRH) stimulates calcium mobilization and influx in pituitary gonadotrophs, and agonist-induced calcium entry through voltage-sensitive channels (VSCC) is required for the maintenance of gonadotropin secretion. However, prolonged or frequent exposure to GnRH attenuates the extracellular Ca2+-dependent cytosolic Ca2+ signal and diminishes hormone secretion. Measurements of membrane Ca2+ currents revealed significant impairment of VSCC activity in gonadotrophs during desensitization by GnRH. VSSC were also inactivated in a calcium-dependent manner during exposure to high K+. Prolonged inactivation of such Ca2+ channels by high K+ reduced the calcium and secretory responses to GnRH and vice versa. The calcium-dependent inactivation of VSCC during GnRH action appears to be a primary factor in the onset of desensitization in pituitary gonadotrophs. This mechanism could also account for the development of agonist-induced refractoriness in other calcium-regulated target cells.     

Influence of Temocapril on Cultured Ventral Spinal Cord Neurons

Temocapril, a angiotensin-converting enzyme (ACE) inhibitor, was tested for neurotrophic activity in primary explant cultures of ventral spinal cord of fetal rats (VSCC). Temocapril had a remarkable effect on neurite outgrowth with a 4.2- to 5.1-fold increased over that of control VSCC at their effective concentrations. In temocapril-treated VSCC, choline acetyltransferase (ChAT) activity was also increased 2.4–3.2 times over that of control at 10^–9 and 10^–8 M, respectively. Our data suggest that temocapril is a candidate for neurotrophic factors on spinal motor neurons in vitro. A possible therapeutic role for temocapril in damaged motor neurons, such as in motor neuropathy and amyotrophic lateral sclerosis, remains to be defined.     

Regulation of calcium channels in brain: implications for the clinical neurosciences

Calcium is a major second messenger in neurons and modulates many neuronal functions, including protein phosphorylation, phospholipid metabolism, cytoskeletal activity, and neurotransmitter release. These important events, which regulate neuronal activity, are directly dependent on the influx of extracellular calcium through voltage-sensitive calcium channels (VSCCs) in the neuronal membrane. Modulation of VSCC function represents an important strategy for regulating neuronal excitability. Although substantial evidence supports the ability of dihydropyridines to block VSCCs and contractility in cardiovascular tissue, their ability to block the majority of neuronal VSCCs remains controversial. Benzodiazepines, and other anticonvulsants, block depolarization-dependent 45Ca uptake through VSCCs in brain synaptosome preparations. In addition, benzodiazepines reduce voltage-gated calcium conductance as determined by voltage clamp studies of identified invertebrate neurons. Inhibition of VSCC activity may be an important mechanism by which these compounds produce their anticonvulsant and sedative effects. Intrasomal injection of calcium-calmodulin-dependent protein kinase modulates calcium conductance in invertebrate neurons, suggesting that protein phosphorylation may be an endogenous regulatory mechanism of VSCC activity. Developing novel pharmacological approaches to regulating VSCCs and understanding the endogenous regulatory mechanisms may lead to new therapeutic approaches to the treatment of neurological diseases.     

凝集素受体在外阴良、恶性肿瘤中的表达

目的探讨细胞表面糖基的变化与外阴表皮细胞良性或恶性增殖的关系。方法采用链霉素抗生物素-过氧化物酶组织化学技术,应用12种凝集素研究60例外阴良、恶性疾病中细胞膜糖基的表达和分布情况。结果从正常外阴表皮到外阴鳞状细胞癌凝集素MPA,ConA,WGA和RCA表达分布无明显的变化。凝集素DBA,LTA和SBA表达均阴性。仅凝集素UEA-1,PNA,VVA,DSA和HPA的表达发生改变。在正常外阴表皮的基底细胞UEA-1,PNA和HPA染色呈阴性,而在外阴鳞状细胞癌出现UEA-1和PNA的阳性表达。在外阴正常表皮VVA和DSA均无表达,但在外阴鳞状细胞增生,外阴尖锐湿疣和外阴鳞状细胞癌中出现不同程度和分布的表达。结论岩澡糖是外阴角朊细胞终末分化的一种标志物;乙酰氨基半乳糖(GalNAc)残基随着外阴表皮细胞的分化而逐渐出现;在外阴细胞恶性转化过程中出现Tn和T抗原的表达;凝集素UEA,VVA,PNA和HPA是外阴表皮良性或恶性增殖的一种好的组织标志物。     

Vasopressin-induced vasoconstriction: two concentration-dependent signaling pathways.

Current scientific literature generally attributes the vasoconstrictor effects of [Arg(8)]vasopressin (AVP) to the activation of phospholipase C (PLC) and consequent release of Ca(2+) from the sarcoplasmic reticulum. However, half-maximal activation of PLC requires nanomolar concentrations of AVP, whereas vasoconstriction occurs when circulating concentrations of AVP are orders of magnitude lower. Using cultured vascular smooth muscle cells, we previously identified a novel Ca(2+) signaling pathway activated by 10-100 pM AVP. This pathway is distinguished from the PLC pathway by its dependence on protein kinase C (PKC) and L-type voltage-sensitive Ca(2+) channels (VSCC). In the present study, we used isolated, pressurized rat mesenteric arteries to examine the contributions of these different Ca(2+) signaling mechanisms to AVP-induced vasoconstriction. AVP (10(-14)-10(-6) M) induced a concentration-dependent constriction of arteries that was reversible with a V(1a) vasopressin receptor antagonist. Half-maximal vasoconstriction at 30 pM AVP was prevented by blockade of VSCC with verapamil (10 microM) or by PKC inhibition with calphostin-C (250 nM) or Ro-31-8220 (1 microM). In contrast, acute vasoconstriction induced by 10 nM AVP (maximal) was insensitive to blockade of VSCC or PKC inhibition. However, after 30 min, the remaining vasoconstriction induced by 10 nM AVP was partially dependent on PKC activation and almost fully dependent on VSCC. These results suggest that different Ca(2+) signaling mechanisms contribute to AVP-induced vasoconstriction over different ranges of AVP concentration. Vasoconstrictor actions of AVP, at concentrations of AVP found within the systemic circulation, utilize a Ca(2+) signaling pathway that is dependent on PKC activation and can be inhibited by Ca(2+) channel blockers.     

The marine toxin okadaic acid is a potent neurotoxin for cultured cerebellar neurons.

The tumor promoter okadaic acid (OKA), is a marine toxin of algal origin, identified as a potent inhibitor of protein phosphatases 1 and 2A, and possibly enhancing calcium influx through voltage dependent calcium channels (VSSC). We now report that OKA at concentrations as low as 0.5 nM produced neurotoxicity, characterized first by the desintegration of the neurites and swelling of cell bodies, and later by cellular death. Non-neuronal cells viability and morphology were unaffected up to at least 5 nM OKA. Neurons sensitivity to the toxin changed with age in culture. Maximum neurotoxicity was observed in neurons at 9 DIC, when the OKA concentration producing half of the maximum reduction in neuronal survival (EC50) was approximately 0.65 nM. At 5 DIC or 19 DIC (EC50 approximately 2.5 nM and approximately 4.5 nM respectively), neurons appeared to be less sensitive to OKA. Neurotoxicity by OKA was not reduced by VSCC antagonists such as nifedipine and verapamil, nor by antagonists of excitatory aminoacid (EAA) receptors including APV, MK801 or CNQX. VSCC antagonists and EAA receptors antagonists fully protected from neurotoxicity induced by depolarization with KCl. These results suggest that OKA mechanism of neurotoxicity may not directly involve VSCC, endogenous EAA release and EAA receptors, but may depend upon other neurochemical events.     

Structure-activity relationships of omega-conotoxins MVIIA, MVIIC and 14 loop splice hybrids at N and P/Q-type calcium channels.

The omega-conotoxins are a set of structurally related, four-loop, six cysteine containing peptides, that have a range of selectivities for different subtypes of the voltage-sensitive calcium channel (VSCC). To investigate the basis of the selectivity displayed by these peptides, we have studied the binding affinities of two naturally occurring omega-conotoxins, MVIIA and MVIIC and a series of 14 MVIIA/MVIIC loop hybrids using radioligand binding assays for N and P/Q-type Ca2+channels in rat brain tissue. A selectivity profile was developed from the ratio of relative potencies at N-type VSCCs (using [125I]GVIA radioligand binding assays) and P/Q-type VSCCs (using [125I]MVIIC radioligand binding assays). In these peptides, loops 2 and 4 make the greatest contribution to VSCC subtype selectivity, while the effects of loops 1 and 3 are negligible. Peptides with homogenous combinations of loop 2 and 4 display clear selectivity preferences, while those with heterogeneous combinations of loops 2 and 4 are less discriminatory. 1H NMR spectroscopy revealed that the global folds of MVIIA, MVIIC and the 14 loop hybrid peptides were similar; however, several differences in local structure were identified. Based on the binding data and the 3D structures of MVIIA, GVIA and MVIIC, we have developed a preliminary pharmacophore based on the omega-conotoxin residues most likely to interact with the N-type VSCC.     

A new omega-conotoxin that targets N-type voltage-sensitive calcium channels with unusual specificity.

A new specific voltage-sensitive calcium channel (VSCC) blocker has been isolated from the venom of the fish-hunting cone snail Conus consors. This peptide, named omega-Ctx CNVIIA, consists of 27 amino acid residues folded by 3 disulfide bridges. Interestingly, loop 4, which is supposed to be crucial for selectivity, shows an unusual sequence (SSSKGR). The synthesis of the linear peptide was performed using the Fmoc strategy, and the correct folding was achieved in the presence of guanidinium chloride, potassium buffer, and reduced/oxidized glutathione at 4 degrees C for 3 days. Both synthetic and native toxin caused an intense shaking activity, characteristic of omega-conotoxins targeting N-type VSCC when injected intracerebroventricularly to mice. Binding studies on rat brain synaptosomes revealed that the radioiodinated omega-Ctx CNVIIA specifically and reversibly binds to high-affinity sites with a K(d) of 36.3 pM. Its binding is competitive with omega-Ctx MVIIA at low concentration (K(i) = 2 pM). Moreover, omega-Ctx CNVIIA exhibits a clear selectivity for N-type VSCCs versus P/Q-type VSCCs targeted respectively by radioiodinated omega-Ctx GVIA and omega-Ctx MVIIC. Although omega-Ctx CNVIIA clearly blocked N-type Ca(2+) current in chromaffin cells, this toxin did not inhibit acetylcholine release evoked by nerve stimuli at the frog neuromuscular junction, in marked contrast to omega-Ctx GVIA. omega-Ctx CNVIIA thus represents a new selective tool for blocking N-type VSCC that displays a unique pharmacological profile and highlights the diversity of voltage-sensitive Ca(2+) channels in the animal kingdom.     

Galanin Reduces Carbachol Stimulation of Phosphoinositide Turnover in Rat Ventral Hippocampus by Lowering Ca2+ Influx Through Voltage-Sensitive Ca2+ Channels

The 29-amino-acid peptide galanin (GAL) caused concentration-dependent inhibition of the accumulation of 3H-inositol phosphates (3H-InsPs) induced by the muscarinic agonist carbachol (CARB; 10(-3)-10(-5) M) in the presence of 5 mM lithium, specifically in tissue miniprisms from rat ventral hippocampus. The inhibitory effect of GAL involved the mono-, bis-, tris-, and tetrakisphosphates formed during activation for 2 min of phospholipase C by CARB (1 mM) in the absence of lithium. GAL (1 microM) did not affect alpha-adrenergic or serotonergic type 2 receptor-mediated phosphoinositide (PI) breakdown in the same tissue. GAL by itself neither acted on basal levels of 3H-InsPs nor affected muscarinic receptors in binding studies. Blockade of the T-, N-, and L-types of voltage-sensitive calcium channel (VSCC) with 200 microM Cd2+ reduced muscarinic receptor-mediated PI breakdown by 50% and abolished the inhibitory effect of GAL (1 microM). Reduction of the extracellular Ca2+ concentration from 1.3 mM to 0.49 microM abolished the GAL inhibition of CARB-stimulated PI hydrolysis. Ca2+ influx promoted by 18 mM K+ depolarization or by 1 microM Bay K 8644, a selective agonist of the L-type VSCC, prevented the inhibitory effect of GAL. Blockade of the L-type VSCC with nifedipine (1 microM) potentiated the inhibitory effects of GAL without affecting muscarinic stimulation of PI breakdown.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of voltage-sensitive calcium channels in a clonal pituitary cell line

We have pharmacologically characterized voltage sensitive calcium channels (VSCCs) in GH3 cells, an anterior pituitary clonal cell line known to secrete prolactin and growth hormone. Raising the medium K+ concentration from 5 to 50 mM caused an immediate increase in net 45Ca2+ uptake which remained apparent over a 15 minute time course. 45Ca2+ uptake was maximally stimulated nearly 10-fold over basal levels. This K+-induced stimulation of Ca2+ uptake was not prevented by 10-5M tetrodotoxin or by replacing sodium with choline in the assay medium. Ca2+ uptake was, however, inhibited by several VSCC antagonists: nitrendipine, D-600, diltiazem and Cd2+. Further, the novel dihydropyridine VSCC agonists, BAY K8644 and CGP 28392, enhanced 50 mM K+-stimulated 45Ca2+ uptake and these effects were blocked by nitrendipine.     

Calcium mobilization and influx during the biphasic cytosolic calcium and secretory responses in agonist-stimulated pituitary gonadotrophs

Stimulation of enriched pituitary gonadotrophs by gonadotropin-releasing hormone (GnRH) elicits dose-dependent biphasic elevations of cytosolic calcium ([Ca2+]i) and luteinizing hormone (LH) release, with rapid initial peaks followed by sustained plateaus during continued exposure to the agonist. A potent GnRH-antagonist, [N-acetyl-D-p-Cl-Phe1,2,D-Trp3,D-Lys6,D-Ala10]GnRH, prevented the biphasic [Ca2+]i and LH responses when added before GnRH, and rapidly abolished both responses to GnRH when added during the plateau phase. In low Ca2+ medium the LH peak responses to GnRH were reduced and the subsequent sustained responses were almost completely abolished; reduction of extracellular Ca2+ during exposure to GnRH caused a prompt decline of LH release. The initial [Ca2+]i peak is derived largely from intracellular calcium mobilization with a partial contribution from calcium influx, while the sustained phase is dependent on the entry of extracellular Ca2+ through both L-type and dihydropyridine-insensitive channels. The presence of L-type voltage-sensitive calcium channels (VSCC) in pituitary gonadotrophs was indicated by the ability of elevated extracellular [K+] to stimulate calcium influx and LH release, and the sensitivity of these responses to dihydropyridine agonist and antagonist analogs. In cells pretreated with high [K+], the peak [Ca2+]i response to GnRH was enhanced but the subsequent plateau phase was markedly attenuated. This divergent effect of sustained membrane depolarization on the biphasic [Ca2+]i response suggests that calcium entry through VSCC initially potentiates agonist-induced mobilization of Ca2+ from intracellular storage sites. However, established Ca2+ entry through depolarization-activated VSCC cannot be further increased by agonist stimulation because both processes operate through the same channels, probably by changes in their activation-inactivation kinetics. Finally, the reciprocal potentiation by the dihydropyridine agonist, BK 8644, and GnRH of [Ca2+]i and LH responses confirms that both compounds act on the same type of channels, i.e., L-type VSCC, that participate in agonist-mediated calcium influx and gonadotropin secretion.