Calcium channels are essential for excitation-contraction coupling and pacemaker activity in cardiac myocytes. While L-type Ca2+ channels (LCC) have been extensively studied, functional roles of T-type channels (TCC) in native cardiac myocytes are still debatable. TCC are activated at more negative membrane potentials than LCC and therefore facilitate slow diastolic depolarization in sinoatrial node cells. Recent studies showed that selective inhibition of TCC produced a marked slowing of the pacemaker rhythm, indicating that contribution of TCC to cardiac automaticity was relatively larger than what had been speculated in previous studies. To re-evaluate TCC, we measured current density and kinetics of TCC in sinoatrial node cells of various mammalian species. Current density of TCC was larger in mice and guinea pigs than in rabbit and porcine sinoatrial node cells. Interestingly, few or no obvious TCC were recorded in porcine sinoatrial node cells. Furthermore, it was demonstrated that TCC could be enhanced by several vasoactive substances, thereby increasing spontaneous firing rate of sinoatrial node cells. TCC may, at least in part, account for different heart rates among various mammalian species. In addition, TCC might be involved in physiological and/or pathophysiological modulations of the heart rate.
Re-expression of fetal genes has been considered to underlie ionic remodeling in diseased heart. T-type Ca2+ channels have been reported to be functionally expressed in embryonic hearts. In this review, we summarize developmental changes of T-type Ca2+ channels in mouse ventricles from 9.5 days postcoitum (dpc) to adulthood, using patch clamp and quantitative PCR. In addition, we introduced T-type Ca2+ channel expression in hypertrophied ventricles caused by myocardial infarction (MI) and aortic banding (AOB). Substantial T-type Ca2+ channel current was recorded at both 9.5 and 18 dpc. The currents were inhibited by Ni2+ at low concentrations. The current was not detectable in the adult stage. Cav3.2 (α1H) mRNA is expressed dominantly at both 9.5 and 18 dpc. Cav3.1 (α1G) increases from 9.5 to 18 dpc, but remains at low level compared with Cav3.2. In contrast, Cav3.1 is greater than Cav3.2 at the adult stage. In MI, Cav3.1 mRNA correlates negatively with brain natriuretic peptide (BNP) mRNA, whereas Cav3.2 mRNA correlates positively with BNP mRNA. In AOB, these correlations are weak. We also analyzed the neuron-restrictive silencer factor (NRSF) in these hearts because it is the suppressor of transcription of the fetal cardiac gene program. The negative correlation between NRSF and BNP was stronger in MI than in AOB. Our findings show that Cav3.2 underlies the functional T-type Ca2+ channel in embryonic heart and suggest that NRSF may regulate Cav3.2 expression in diseased hearts.
Expression of T-type Ca2+ current in the ventricle varies during development and in cardiac diseases. The alteration in quantity of two isoforms of T-type Ca2+ channel genes in the heart, CACNA1G and CACNA1H, contributes to the changes of T-type Ca2+ channel activity. However, the precise mechanisms governing the transcription of T-type Ca2+ channel genes remain largely unknown. In this review, we briefly describe our recent finding that a transcriptional repressor named neuron-restrictive silencer factor is a potent regulator of T-type Ca2+ channel gene expression.
T-type Ca2+ channels are present in cardiovascular, neuronal, and endocrine systems; and they are now receiving attention as novel therapeutic targets. Many drugs and compounds non-specificaly block T-type Ca2+ channels. Certain dihydropyridine compounds, such as efonidipine, have blocking activity on both L-type and T-type Ca2+ channels which possibly underlies their excellent clinical profiles such as minimum reflex tachycardia and renal protection. Selective inhibitors of T-type Ca2+ channels, such as non-hydrolyzable mibefradil and R(−)-efonidipine, are powerful pharmacological tools for further studies and may lead to the development of novel therapeutic strategies.
Since conventional Ca2+ antagonists, with predominant blockade of L-type voltage-dependent Ca2+ channels, elicit preferential dilation of afferent arterioles, they might ostensibly aggravate glomerular hypertension. Recently, novel Ca2+ antagonists, with inhibitory action on L-/T-type Ca2+ channels, have been reported to dilate both afferent and efferent arterioles. The present review attempted to characterize the renal action of these Ca2+ antagonists and evaluated the consequences following the treatment with these agents. In contrast to conventional Ca2+ antagonists (e.g., nifedipine), novel antagonists (e.g., benidipine, efonidipine) potently dilated afferent and efferent arterioles; their action on efferent arterioles appeared to be mediated by the T-type Ca2+ channel blockade, probably through the inhibition of the intracellular Ca 2+ release. The comparison of the anti-proteinuric action in subtotally nephrectomized rats showed that efonidipine exerted more prominent action than nifedipine. Furthermore, Ca2+ antagonists with T-type Ca2+ inhibitory action inhibited renin/aldosterone release and proinflammatory process. Finally, patients with chronic renal disease given a 48-week efonidipine treatment showed reduced proteinuria, and this effect was seen even when mean arterial blood pressure failed to become less than 100 mmHg. Collectively, T-type Ca2+ channel blockade provides beneficial action in renal injury. Various mechanisms serve to protect against renal injury, including systemic/glomerular hemodynamic action and non-hemodynamic mechanisms.
Effects of substance P (SP) and selective tachykinin agonists on neurotransmission at guinea-pig intracardiac ganglia were studied in vitro. Voltage responses of neurons to superfused tachykinins and nerve stimulation were measured using intracellular microelectrodes. Predominant effects of SP (1 μM) were to cause slow depolarization and enable synaptic transmission at low intensities of nerve stimulation. Augmented response to nerve stimulation occurred with 29 of 40 intracardiac neurons (approx. 73%). SP inhibited synaptic transmission at 23% of intracardiac neurons but also caused slow depolarization. Activation of NK3 receptors with 100 nM [MePhe7]neurokinin B caused slow depolarization, enhanced the response of many intracardiac neurons to low intensity nerve stimulation or local application of acetylcholine, and triggered action potentials independent of other stimuli in 6 of 42 neurons. The NK1 agonist [Sar9,Met(O2)11]SP had similar actions but was less effective and did not trigger action potentials independently. Neither selective agonist inhibited cholinergic neurotransmission. We conclude that SP can function as a positive or negative neuromodulator at intracardiac ganglion cells, which could be either efferent neurons or interneurons. Potentiation occurs primarily through NK3 receptors and may enable neuronal responses with less preganglionic nerve activity. Inhibition of neurotransmission by SP is most likely explained by the known blocking action of this peptide at ganglionic nicotine receptors.
The biological basis for the therapeutic mechanisms of depression are still unknown. While performing EST (expressed sequence tag) analysis to identify some molecular machinery responsible for the antidepressant effect, we determined the full-length nucleotide sequence of rat frizzled-3 protein (Frz3) cDNA. Interestingly, Northern blot analysis demonstrated that elevated levels of Frz3 were expressed continually from embryonic day 20.5 to postnatal 4 weeks in developing rat brain. In adult rat brain, Frz3 mRNA was expressed predominantly in the cerebral cortex and hypothalamus and moderately in the hippocampus. Using real-time quantitative PCR, we demonstrated that chronic treatment with two different classes of antidepressants, imipramine and sertraline, reduced Frz3 mRNA expression significantly in rat frontal cortex. Electroconvulsive treatment (ECT) also reduced Frz3 expression. In contrast, antidepressants and ECT failed to reduce Frz2 expression. Additionally, chronic treatment with the antipsychotic drug haloperidol did not affect Frz3 expression. Recently, the Frz/Wingless protein pathway has been proposed to direct a complex behavioral phenomenon. In conclusion, the Frz3-mediated signaling cascade may be a component of the molecular machinery targeted by therapeutics commonly used to treat depression.
Natto, steamed soybeans fermented by Bacillus subtilis natto, is a traditional Japanese food. We derived a purified protein layer, called NKCP as a trade mark, from B. subtilis natto fermentation. In the present study, we examined the fibrinolytic and antithrombotic effects of NKCP and identified its active component to clarify the fibrinolytic effect of NKCP observed in preliminary clinical trials previously. The active component of NKCP was identified as a 34-kilodalton protein designated bacillopeptidase F. NKCP showed direct degradation of artificial blood clot in saline. The protease activity, accounting for the fibrinolytic effect of NKCP, was examined with a chromogenic substrate for plasmin. Dose-dependent prolongations of both prothrombin time and active partial thromboplastin time were observed in rats with intra-duodenum administration of NKCP. Our in vitro and in vivo studies suggest that NKCP has both a fibrinolytic effect and an antithrombotic effect similar to heparin. Because NKCP is derived from food and has safety data demonstrated by previous animal experiments and preliminary clinical trials, NKCP is considered as safe for clinical use.
Endothelial cell (EC) barrier dysfunction is associated with many types of vascular diseases. Investigators have hypothesized that altered EC contraction in conjunction with morphological changes may lead to EC dysfunction. However, the nature of EC contraction and its regulatory mechanisms are not fully understood. In this study we measured thrombin-induced force in bovine arterial EC force using EC fibers that were grown in a collagen matrix. Contraction, which occurred in time- and dose-dependent fashion, was elicited by thrombin. The thrombin-enhanced EC layer permeability was correlated with EC fiber contraction. These results suggest that EC contractile response is involved in alteration of EC barrier function. During the initial period of thrombin stimulation, cadherin complexes were disrupted and cell-to-cell connections were reduced. This was dependent on the transient increase in intracellular calcium concentration and myosin phosphorylation. Rho kinase activation led to rearrangement of actin stress fibers (ASF). Paracellular holes were created in the EC layer in parallel to EC morphological change. Our findings suggest that EC layer permeability is regulated by two distinguishable steps. In the initial period, the cell-to-cell connection was reduced in a calcium-dependent fashion. Subsequently, Rho kinase and ASF-mediated force development increased EC layer permeability via morphological change of EC.
Adrenomedullin (AM) is a hypotensive peptide that belongs to a family of peptides structurally related to calcitonin gene-related peptide (CGRP). The present study examined the effect of AM on adrenergic nerve-mediated vasoconstriction in rat perfused mesenteric vascular beds without endothelium. Perfusion of AM at 0.1 nM but not 10 nM increased vasoconstrictor responses to periarterial nerve stimulation (PNS) (1 – 4 Hz), while AM at 10 nM significantly attenuated vasoconstriction induced by bolus injection of norepinephrine (NE). In preparations treated with capsaicin (a CGRP depletor), pressor responses to both PNS and NE injection were markedly attenuated by AM. Perfusion of CGRP(8 – 37) (a CGRP-receptor antagonist) significantly potentiated the PNS- but not the NE-induced vasoconstriction. Combined perfusion of CGRP(8 – 37) and AM had no effect on the PNS-induced response and antagonized the inhibitory effect of AM on the NE-induced response. AM(22 – 52) (an AM-receptor antagonist) did not influence the effect of AM. These findings suggest that AM facilitates adrenergic vasoconstriction by inhibiting neurotransmission of CGRP-containing nerves, which counteract adrenergic nerve-mediated vasoconstriction.
The effects of indomethacin and nabumetone on urine and electrolyte excretion in conscious rats were examined. Male Sprague-Dawley rats were housed individually for a five-week duration, consisting of acclimatization, control, experimental, and recovery phases. During the experimental phase, rats were given either indomethacin (1.5 mg · kg−1 body weight · day−1 in 0.5 ml saline, n = 10), nabumetone (15 mg · kg−1 body weight · day−1 0.5 ml saline, n = 10), or 0.5 ml saline alone (n = 10) for a period of two weeks. Water and food intake, body weight, urine output, and electrolyte excretions were estimated. Data were analyzed using two-way ANOVA. Urine output in the indomethacin- and nabumetone-treated groups was not different from the controls, but was significantly different between the drug-treated groups (P<0.01). Sodium, potassium, calcium, and magnesium excretions were not different between nabumetone-treated and control rats. However, sodium and potassium excretion was significantly lower in rats receiving indomethacin when compared to the control rats. Calcium and magnesium outputs, although did not differ from the controls, nevertheless decreased significantly with indomethacin (P<0.01). It appears that indomethacin and nabumetone when given at maximum human therapeutic doses may affect urine and electrolyte output in conscious rats.
Nerve growth factor (NGF) is an important substance in the skin, where it can modulate nerve maintenance and repair. However, the direct link between NGF and pruritic disease such as atopic dermatitis is not yet fully understood. To determine whether NGF plays a major role in atopic dermatitis and in the development or maintenance of skin lesions, we performed a study using NC/Nga mice and compared mice with and without skin lesions. Our examinations of the NC/Nga mice sought to detect nerve fibers in the epidermis, measured serum and skin NGF content, and observed skin NGF by immunohistochemistry staining. We also examined the effects of anti-NGF antibody on dermatitis symptoms in NC/Nga mice. In these mice, nerve fibers were significantly increased in the epidermis of lesioned skin, and the NGF content of the serum and skin was significantly elevated. Anti-NGF antibodies significantly inhibited the development and proliferation of skin lesions and epidermal innervation and significantly inhibited any growth in scratching but did not ameliorate scratching already developed. Our findings suggest that NGF plays important roles in the pathogenesis of atopic dermatitis-like skin lesions and that inhibiting the physiological effects of NGF or suppressing increased NGF production may prevent or even moderate the symptoms of atopic dermatitis.
The aim of this study was to investigate the disposal of aggregated protein in the glomeruli of spontaneously diabetic mice. Diabetic mice, KK-Ay and db/db, and age-matched ICR mice were injected intravenously with aggregated bovine serum albumin (a-BSA) at 0.6 mg/g, and the glomeruli and the blood were obtained. Diabetic mice had larger amounts of a-BSA in their glomeruli than the ICR mice, threefold in KK-Ay and twofold in db/db, at 3 h after the a-BSA injection. Additionally, the disappearance of a-BSA was retarded in the diabetic glomeruli. KK-Ay displayed a-BSA in the glomeruli 24 h after the a-BSA injection and db/db did after 12 h, while the ICR did by 8 h. In spite of increases of insulin to similar degrees in both strains of diabetic mice after the a-BSA injection, blood glucose levels markedly decreased in KK-Ay compared with db/db. There were no histopathological alterations in the glomeruli of the diabetic mice. Depositions of a-BSA were confirmed to be higher in the diabetic glomeruli by the immunofluoresnce technique, and KK-Ay displayed higher depositions of a-BSA than did db/db. The present study suggests that hyperglycemia is involved in the increased deposition of aggregated protein in the glomeruli and that the degradation of aggregated protein is retarded in diabetic glomeruli.
Polygonum multiflorum stilbeneglycoside (PMS) is a water-soluble fraction of Polygonum multiflorum Thunb., one of the most famous tonic traditional Chinese medicines, that has protective effects on the cardiovascular system. The purpose of the present study is to elucidate the effects of PMS on macrophage-derived foam cell functions and the reduction of severity of atherosclerosis in hypercholesterolemic New Zealand White (NZW) rabbits. NZW rabbits were fed for 12 weeks with a normal diet, a high cholesterol diet, or a high cholesterol diet associated with irrigation with different doses of PMS (25, 50, or 100 mg/kg). Treatment of NZW rabbits fed with high cholesterol diet with 100 mg/kg PMS attenuated the increase in plasma cholesterol, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, and plasma triglyceride. Treatment with 50 and 100 mg/kg PMS caused 43% and 60% decrease in atherosclerotic lesioned area ratio to total surface area, respectively. In U937 foam cells, PMS could decrease the high expression of intercellular adhesion molecule (ICAM)-1 protein and the vascular endothelial growth factor (VEGF) protein levels in the medium induced by oxidized lipoprotein when analyzed by flow cytometry. The results proved that PMS is a powerful agent against atherosclerosis and that PMS action could possibly be through the inhibition of the expression of ICAM-1 and VEGF in foam cells.