Sex- and afferent-specific differences in histamine receptor expression in vagal afferents of rats: A potential mechanism for sexual dimorphism in prevalence and severity of asthma.

The incidence of asthma is more common in boys than in girls during the childhood, and more common in premenopausal female than age-matched males. Our previous study demonstrated a gender difference in histamine-mediated neuroexcitability in nodose ganglia neurons (NGNs), highlighting a possibility of histamine-mediated gender difference in asthma via visceral afferent function. In the present study, we aimed to explore the gender difference in expression profiles of histamine receptors (HRs) in nodose ganglia (NG) and individual identified NGNs to provide deeper insights into the mechanisms involved in sexual dimorphism of asthma. Western-blot and SYBR green RT-PCR showed that H2R and H3R were highly expressed in NG of females compared with males and downregulated in ovariectomized females. H1R was equally expressed in NG of both sexes and not altered by ovariectomy. Furthermore, this highly expressive H2R and H3R were distributed in both myelinated and unmyelinated NGNs isolated from adult female rats by immunofluorescence and single-cell RT-PCR. H3R widely distributed in all tested neuron subtypes and its expression did not show significant difference among neuron subtypes. H2R was widely and highly expressed in low-threshold and sex-specific subpopulation of myelinated Ah-types compared with myelinated A- and unmyelinated C-type NGNs. Unexpectedly, weak expression of H1R was detected in both myelinated and unmyelinated NGNs by immunofluorescence, which was further confirmed by single-cell RT-PCR. Our results suggest that the sexual dimorphism in the expression of H2R and H3R in vagal afferents very likely contributes, at least partially, to the gender difference in prevalence and severity of asthma.

Antagonistic effects of berbamine on [Ca2+]i mobilization by KCl, norepinephrine, and caffeine in newborn rat cardiomyocytes.

AIM: To study the effects of berbamine (Ber) on intracellular calcium concentration ([Ca2+]i) mobilized by KCl depolarization, norepinephrine (NE), and caffeine. METHODS: [Ca2+]i was measured with fluorescent intensity (FI) by confocal microscope in single cultured cardiomyocytes of newborn rats loaded with Fluo 3-AM 2 mumol.L-1. RESULTS: FI value of [Ca2+]i in control level was 248 +/- 70 in the presence of extracellular calcium 1.5 mmol.L-1 and was not changed by Ber 3-30 mumol.L-1. KCl (60 mmol.L-1)- and NE (30 mumol.L-1)-induced [Ca2+]i mobilizations were inhibited (P < 0.01) by Ber 30 mumol.L-1, similar to that of verapamil (Ver). The inhibitory effect of Ber on [Ca2+]i induced by KCl was further increased (P < 0.05) in the presence of egtazic acid 3 mmol.L-1, but that on [Ca2+]i induced by NE was not changed. The [Ca2+]i mobilized by caffeine 80 and 160 mumol.L-1 in D-Hanks' solution was not affected (P > 0.05) by Ber and Ver. CONCLUSION: Ber possessed the antagonistic effects on [Ca2+]i increases via voltage-dependent Ca2+ channel and receptor-operated Ca2+ channel in newborn rat cardiomyocytes, but without effect on intracellular Ca2+ release.

Cytosolic-Ca2+ and coxsackievirus B3-induced apoptosis in cultured cardiomyocytes of rats.

AIM: To explore the role of cytosolic free calcium ([Ca2+]i) in apoptosis induced by coxsackievirus B3 (CVB3) in cultured cardiomyocytes of rats. METHODS: Primary cultured cardiomyocyte was prepared from Wistar rats ages 2-3 d. The apoptosis in cardiomyocyte was determined by terminated deoxynucleotide transferase directed d-UTP nick and end labeling (TUNEL) method, and the apoptosis was observed under a transmission electron microscope. [Ca2+]i in single cardiomyocyte loaded with Fluo 3-AM was measured by confocal microsorope. RESULTS: (1) The concentration of CVB3 in the medium reached the peak at 24 h after CVB3 infection. (2) The apoptotic cells were not found in CVB3-infected cardiomyocyte in first 10 h, but amounted to 5% at 17 h, 60% at 24 h, and 90% at 36 h. (3) The peak value of [Ca2+]i elevation reached at 17 h after CVB3 infection (P < 0.01). (4) The characteristics of apoptosis was also seen by transmission electron microscope. CONCLUSION: CVB3 induced the apoptosis in cultured cardiomyocyte, and [Ca2+]i mobilization was involved in the signal transduction process in apoptosis cells, and played an important role especially in the early stage of apoptosis induced by CVB3.

Effects of berbamine on ATP-induced [Ca2+]i mobilization in cultured vascular smooth muscle cells and cardiomyocytes.

AIM: To study the effects of berbamine (Ber) on [Ca2+]i homeostasis induced by adenosine triphosphate (ATP) in vascular smooth muscle cells (VSMC) of rabbits and cardiomyocytes of rats. METHODS: Both cell types were cultured and loaded with Fura 3-AM. [Ca2+]i was measured by fluorescent intensity (FI) in each cell with confocal microscopy. RESULTS: (1) ATP 30 mumol.L-1 elevated [Ca2+]i in VSMC and cardiomyocytes, FI values reached 660 +/- 258 and 1058 +/- 252 from 250 +/- 84 and 218 +/- 76 at 19 s +/- 5 s and 11.8 s +/- 2.4 s, but FI in nucleus was not changed in VSMC. (2) Ber 30 mumol.L-1 did not affect the resting FI in both cell types, but prolonged the time to peak (P < 0.01) and reduced the FI elevated by ATP (P < 0.01), but not completely inhibited even at 100 mumol.L-1. (3) In D-Hanks' solution or in the presence of egtazic acid (EGTA) 3 mmol.L-1, the inhibitory effect of Ber was not seen (P > 0.05). (4) All effects of Ber on ATP-induced [Ca2+]i mobilization were similar to those of Ver 10 mumol.L-1. CONCLUSION: In VSMC and cardiomyocytes, ATP-induced CA2+ influx was inhibited by Ber and Ver, while the Ca2+ release was not.

Effects of MK-447 on platelet shape change, aggregation, and ATP release by collagen, ADP, and stable analogue of thromboxane A2 in rabbit platelets.

AIM: To investigate the effects of MK-447 on platelet shape change, aggregation, and ATP release by collagen (Col), ADP, and stable analogue of thromboxane A2 (STA2) in rabbits. METHODS: Platelet shape change and aggregation were quantified in light transmission by turbidimetric method and release reaction was assessed by the amount of ATP in platelet-rich plasma (PRP). RESULTS: (1) MK-447 100-700 mumol.L-1 caused only the shape change, which was not inhibited by indometacin 3 mumol.L-1. Platelet shape changes by Col, ADP, and STA2 were reduced (P < 0.01) after the addition of MK-447. The lag phase was prolonged (P < 0.01) in Col and shortened (P < 0.01) in ADP. (2) MK-447 reduced the aggregation by Col 5 mg.L-1 (P < 0.01), and enhanced that by ADP 0.3-10 mumol.L-1 and STA2 0.1-3 mumol.L-1 (P < 0.01). (3) The release reaction by STA2 1-3 mumol.L-1 was also increased (P < 0.01). The effects of MK-447 on STA2 were not inhibited by S-145. CONCLUSION: MK-447 induced the platelet shape change, and showed the dual effects, inhibition or enhancement, on the actions by different aggregating agents.

Inhibitory effects of ONO-3708 and S-145 on shape change and aggregation of rabbit platelets induced by STA2.

AIM: To study the mode of inhibition of ONO-3708 and S-145, 2 antagonists of thromboxane A2 (TXA2) receptors, against the rabbit platelet shape change and aggregation induced by stable analogue of TXA2 (STA2). METHODS: The platelet shape change and aggregation were quantified by the light transmission through platelet-rich plasma (PRP) and the intracellular calcium concentration ([Ca2+]i) was measured by fluorescence and imaging. RESULTS: (1) In PRP, STA2 (3 mumol.L-1)-induced aggregation was inhibited by egtazic acid 3 mmol.L-1, ONO-3708 300 mumol.L-1, and S-145 1 mumol.L-1 (P < 0.01), but not by indometacin (Ind) 3 mumol.L-1. The shape change induced by STA2 was inhibited only by S-145 in a concentration-dependent manner. S-145 1 and 3 mumol.L-1 were required to inhibit the shape change and aggregation. (2) The inhibitory effect of S-145, but not ONO-3708, was increased along with the prolongation of preincubation. (3) ONO-3708 lost the inhibitory effect on STA2-induced aggregation after washing, while the inhibitory effect of S-145 was enhanced by prolongation of preincubation and remained after washing. (4) STA2 3 mumol.L-1-induced [Ca2+]i mobilization was unaffected by Ind, partially reduced by ONO-3708 and egtazic acid 3 mmol.L-1 (P < 0.01), but completely inhibited by S-145 (P < 0.01). CONCLUSION: S-145 and ONO-3708 were bound to a different site of the TXA2 receptor.