Inositol Phosphate Ca Research Articles

Germline Prokineticin Receptor 2 (PROKR2) Variants Associated With Central Hypogonadism Cause Differental Modulation of Distinct Intracellular Pathways

Defects of prokineticin pathway affect the neuroendocrine control of reproduction, but their role in the pathogenesis of central hypogonadism remains undefined, and the functional impact of the missense PROKR2 variants has been incompletely characterized. In a series of 246 idiopathic central hypogonadism patients, we found three novel (p.V158I, p.V334M, and p.N15TfsX30) and six already known (p.L173R, p.T260M, p.R268C, p.V274D, p.V331M, and p.H20MfsX23) germline variants in the PROKR2 gene. We evaluated the effects of seven missense alterations on two different prokineticin receptor 2 (PROKR2)-dependent pathways: inositol phosphate-Ca(2+) (Gq coupling) and cAMP (Gs coupling). PROKR2 variants were found in 16 patients (6.5%). Expression levels of variants p.V158I and p.V331M were moderately reduced, whereas they were markedly impaired in the remaining cases, except p.V334M, which was significantly overexpressed. The variants p.T260M, p.R268C, and p.V331M showed no remarkable changes in cAMP response (EC50) whereas the IP signaling appeared more profoundly affected. In contrast, cAMP accumulation cannot be stimulated through the p.L173R and p.V274D, but IP EC50 was similar to wt inp.L173R and increased by 10-fold in p.V274D. The variant p.V334M led to a 3-fold increase of EC50 for both cAMP and IP. Our study shows that single PROKR2 missense allelic variants can either affect both signaling pathways differently or selectively. Thus, the integrity of both PROKR2-dependent cAMP and IP signals should be evaluated for a complete functional testing of novel identified allelic variants.

Cytokine upregulation of proteinase-activated-receptors 2 and 4 expression mediated by p38 MAP kinase and inhibitory kappa B kinase beta in human endothelial cells.

Up-regulation of proteinase-activated receptor-2 (PAR2) is a factor in a number of disease states and we have therefore examined the signalling pathways involved in the expression of the receptor. We investigated the effects of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), trypsin and the PAR2 activating peptide, 2-furoyl(2f)-LIGKV-OH on both mRNA and functional expression of PAR2 in human umbilical vein endothelial cells (HUVECs). The effect of specific chemical inhibitors and dominant negative adenovirus constructs of the mitogen-activated protein kinase (MAPK) cascade and the nuclear factor kappa B (NF-kappaB) signalling pathway was assessed. Methods included semi-quantitative and quantitative RT-PCR, [(3)H]inositol phosphate (IP) accumulation and Ca(2+)-dependent fluorescence. The above agonists induced both mRNA and functional expression of PAR2; PAR4 mRNA, but not that for PAR1 or PAR-3, also increased following TNFalpha treatment. Inhibition of p38 MAP kinase reduced PAR2 and PAR4 expression, whilst inhibition of MEK1/ERK/JNK was without effect. A similar dependency upon p38 MAP kinase was observed for the expression of PAR4. TNFalpha -induced enhancement of PAR2 stimulated [(3)H]-inositol phosphate accumulation (IP) and Ca(2+) signalling was abolished following SB203580 pre-treatment. Infection with adenovirus encoding dominant-negative IKKbeta (Ad.IKKbeta(+/-)) and to a lesser extent dominant-negative IKKalpha (Ad.IKKalpha(+/-)), substantially reduced both control and IL-1beta- induced expression of both PAR2 and PAR4 mRNA and enhancement of PAR2-stimulated IP accumulation and Ca(2+) mobilisation. These data reveal for the first time the signalling events involved in the upregulation of both PAR2 and PAR4 during pro-inflammatory challenge.

Inhibition of Bradykinin-Induced Phosphoinositide Hydrolysis and Ca 2+ Mobilisation by Phorbol Ester in Rat Cultured Vascular Smooth Muscle Cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca 2+ concentration ([Ca 2+] i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 μM) for 30 min almost abolished the BK-induced IP formation and Ca 2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC 50) and maximal inhibition of BK induced an increase in [Ca 2+] i, were 7.8 ± 0.3 M and 1 μM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca 2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-α, βI, βII, δ, ϵ, and ζ isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 μM PMA for either 1 or 24 h did not significantly change the K D and B max of the BK receptor for binding (control: K D = 1.7 ± 0.2 nM; B max = 47.3 ± 4.4 fmol mg protein ), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these resuts demonstrate that translocation of PKC-α, βI, βII, δ, ϵ, and ζ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca 2+ mobilisation in VSMCs.

Pharmacological and Functional Characterization of Bradykinin Receptors in Rat Cultured Vascular Smooth Muscle Cells

The pharmacological properties of bradykinin receptors were characterized in rat cultured vascular smooth muscle cells (VSMCs) using [ 3H]-bradykinin as a ligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant ( K D) of 1.2 ± 0.2 nM and a maximum receptor density ( B max) of 47.3 ± 4.4 fmol/mg protein. The specific binding of [ 3H]-bradykinin to VSMCs was inhibited by the B 2 receptor-selective agonists (bradykinin and kallidin) and antagonists ([D-Arg 0, Hyp 3, Thi 5, D-Tic 7, Oic 8]-bradykinin (Hoe 140) and [D-Arg 0, Hyp 3, Thi 5,8, D-Phe 7]-bradykinin) with an order of potency as kallidin = bradykinin = Hoe 140 > [D-Arg 0, Hyp 3, Thi 5,8, D-Phe 7]-bradykinin, but not by a B 1 receptor-selective agonist (des-Arg 9-bradykinin) and antagonist ([Leu 8, des-Arg 9]-bradykinin). Stimulation of VSMCs by bradykinin produced a concentration-dependent inositol phosphate (IP) accumulation, and initial transient peak of [Ca 2+] i with half-maximal responses (pEC 50) were 7.53 and 7.69, respectively. B 2 receptor-selective antagonists (Hoe 140 and [D-Arg 0, Hyp 3, Thi 5,8, D-Phe 7]-bradykinin) significantly antagonized the bradykinin-induced responses with pK B values of 8.3–8.7 and 7.2–7.9, respectively. Pretreatment of VSMCs with pertussis toxin (100 ng/ml, 24 h) did not alter the bradykinin-induced inositol phosphate accumulation and [Ca 2+] i changes in VSMCs. Removal of external Ca 2+ led to a significant attenuation of responses induced by bradykinin. Influx of external Ca 2+ was required for the bradykinin-induced responses, since Ca 2+-channel blockers, nifedipine, verapamil, and Ni 2+, partially inhibited the bradykinin-induced IP accumulation and Ca 2+ mobilization. These results demonstrate that bradykinin stimulates phosphoinositide hydrolysis and Ca 2+ mobilization via a pertussis toxin-insensitive G-protein in rat VSMCs. Bradykinin B 2 receptors may be predominantly mediating IP accumulation and subsequently induction of Ca 2+ mobilization may function as the transducing mechanism for bradykinin-stimulated contraction of vascular smooth muscle.

5-Hydroxytryptamine-Induced Phosphoinositide Hydrolysis and Ca 2+ Mobilisation in Canine Cultured Aorta Smooth Muscle Cells

The effect of 5-hydroxytryptamine (5-HT) on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and intracellular Ca 2+ ([Ca 2+] i) changes was investigated in canine cultured aorta smooth muscle cells (ASMCs). 5-HT-stimulated inositol phosphate (IP) accumulation was time and concentration dependent with a half-maximal response (pEC 50) and a maximal response at 6.4 and 10 μM, n = 6, respectively. Stimulation of ASMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation in [Ca 2+] i. The half-maximal response (pEC 50) values of 5-HT for the peak and sustained plateau were 7.1 and 6.9, respectively. Ketanserin and mianserin (1 and 3 nM), 5-HT 2A antagonists, were equipotent and had high affinity in antagonising the 5-HT-induced IP accumulation and [Ca 2+] i change with p K B values of 8.6–9.1 and 8.6–9.4, respectively. In contrast, the concentration-effect curves of 5-HT-induced IP and [Ca 2+] i responses were not shifted until the concentrations of NAN-190 and metoclopramide (5-HT 1A and 5-HT 3 receptor antagonists, respectively) were increased to as high as 1 μM with p K B values of 5.7–6.3 and 6.1–6.6, respectively, indicating that the 5-HT receptor-mediated responses had low affinity for these antagonists. Pre-treatment of ASMCs with pertussis toxin (100 ng/mL, 24 h) caused a significant inhibition of 5-HT-induced IP accumulation and [Ca 2+] i change in ASMCs. Depletion of external Ca 2+ or removal of Ca 2+ by addition of EGTA led to a significant attenuation of IP accumulation and [Ca 2+] i change induced by 5-HT. Influx of external Ca 2+ was required for the 5-HT-induced responses, because Ca 2+-channel blockers—verapamil, nifedipine and Ni 2+—partly inhibited the 5-HT-induced IP accumulation and Ca 2+ mobilisation. The sustained elevation of [Ca 2+] i response to 5-HT was dependent on the presence of external Ca 2+. Removal of external Ca 2+ by addition of 5 mM EGTA during the sustained phase caused a rapid decline in [Ca 2+] i to lower than the resting level. The sustained elevation of [Ca 2+] i could then be evoked by addition of 1.8 mM Ca 2+ in the continued presence of 5-HT. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis and Ca 2+ mobilisation, at least in part, through a pertussis toxin-sensitive G protein in canine ASMCs. 5-HT 2A receptors may be predominantly mediating IP accumulation, and subsequently IP-induced Ca 2+ mobilisation may function as the transducing mechanism for 5-HT-stimulated contraction of aorta smooth muscle.

P1-Purinergic Receptor-Mediated Modulation of TSH Actions on FRTL-5 Thyroid Cells: Possible Switching From cAMP Pathway to Inositol Phosphate-Ca System

Abstract In thyroid cells, a PI-agonist, via G1 like protein, enhanced a TSH-induced I−-efflux by intensifying a TSH-dependent inositol polyphosphate production followed by a Ca2+ mobilization, but diminished a TSH-dependent DNA synthesis by attenuating a TSH-dependent cAMP accumulation.

The four dimensions of calcium signalling in Xenopus oocytes

This review focuses on the inositol phosphate Ca 2+ signalling pathway in Xenopus oocytes. The known characteristics of the individual elements of this cascade — from the membrane receptors to the intracellular Ca 2+ stores — will be covered. Based on this knowledge, a simple model will then try to account for the behaviour of the newly recognized oscillations of free intracellular Ca 2+ and propagated Ca 2+ waves. Finally, some of the potential physiological functions of the inositol phosphate pathway will be summarized. Although there is no systematic attempt to contrast the findings in the oocyte to those in other cells, the readers of this journal will not fail to notice a high degree of similarity. Although this may seem unexciting at first, it suggests that the inositol phosphate signalling pathway may be strikingly conserved across species.