Effects of high extracellular calcium concentrations on phosphoinositide turnover and inositol phosphate metabolism in dispersed bovine parathyroid cells.

We previously showed that high extracellular calcium (Ca2+) concentrations raise the levels of inositol phosphates in bovine parathyroid cells, presumably via the G protein-coupled, "receptor-like" mechanism through which Ca2+ is thought to regulate these cells. To date, however, there are limited data showing Ca(2+)-evoked hydrolysis of phosphoinositides with attendant increases in the levels of the biologically active 1,4,5 isomer of inositol trisphosphate (IP3) that would be predicted to arise from such a receptor-mediated process. In the present studies we used HPLC and TLC, respectively, to quantify the high Ca(2+)-induced changes in various inositol phosphates, including the isomers of IP3, and phosphoinositides in bovine parathyroid cells prelabeled with [3H]inositol. In the absence of lithium, high Ca2+ dose dependently elevated the levels of inositol-1,4,5-trisphosphate [I(1,4,5)P3], with a maximal, 4- to 5-fold increase within 5 s; the levels of inositol 1,3,4-trisphosphate [I(1,3,4)P3] first rose significantly at 5-10 s and remained 5- to 10-fold elevated for at least 30 minutes. These changes were accompanied by reciprocal 29-36% decreases in PIP2 (within 5-10 s, the earliest time points examined), PIP (within 60 s), and PI (within 60 s). These results document that, as in other cells responding to more classic "Ca(2+)-mobilizing" hormones, the high Ca(2+)-evoked increases in inositol phosphates in bovine parathyroid cells arise from the hydrolysis of phosphoinositides, leading to the rapid accumulation of the active isomer of IP3. The latter presumably underlies the concomitant spike in the cytosolic calcium concentration (Ca(i)) in parathyroid cells.     

Effects of fluoride on parathyroid hormone secretion and intracellular second messengers in bovine parathyroid cells

Fluoride ion (F-) alone or in conjunction with aluminum (Al3+) has been shown to stimulate the activity of guanine nucleotide-binding proteins (G proteins) in cell membrane preparations from a variety of cell types and in intact hepatic cells. Several studies have indicated that G proteins are involved in the regulation of parathyroid hormone (PTH) secretion. Intracellular second messengers which modulate PTH secretion (e.g., cAMP) have also been found to be regulated by G proteins. We have, therefore, employed F- as a probe to investigate the possible role of G proteins in the modulation of PTH release and the intracellular second messengers that have been implicated in the control of PTH secretion. F- produces a dose-dependent inhibition of PTH release with a maximal inhibitory effect (67%) at 5 mM. F- exerts its inhibitory effect within 5 min and the degree of suppression of PTH secretion gradually increases over 1 hr. F- (5 mM) inhibits PTH secretion at 0.5 mM Ca2+ to the level observed with 2 mM Ca2+ alone; moreover, the effects of F- and high Ca2+ are not additive. While 1 mM F- suppresses PTH secretion by only 21%, and 10 microM Al3+ has virtually no effect at all, together they inhibit PTH release approximately to the level (63% inhibition) observed with 5 mM NaF alone. In the presence of 10(-5) M dopamine, F- produces a concentration-dependent inhibition of cAMP accumulation (0.684 +/- 0.033 pmoles/10(5) cells at 0 mM F- vs. 0.256 +/- 0.048 at 5 mM F-). However, the F- -induced decrease in cAMP cannot account for the inhibition of PTH release by this agent, since addition of methylisobutylxanthine (10(-4) M) by F- -treated cells raises intracellular cAMP content above that of control cells but fails to reverse the inhibition of PTH release. The cytosolic calcium concentration in Fura-2-loaded cells increases from 210 +/- 20 nM to 340 +/- 44 nM after 5 mM F- was added to incubation media. Prior removal of extracellular Ca2+ by EGTA totally blocks the F- -induced rise in cytosolic Ca2+ without preventing the inhibition of PTH release by NaF. F- also produces a time- and dose-dependent increase in the accumulation of IP, IP2, and IP3 in cells prelabeled with [3H]inositol and incubated with 10 mM Li+, consistent with activation of phospholipase C. We conclude that F- is a potent inhibitor of PTH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Polyarginine, polylysine, and protamine mimic the effects of high extracellular calcium concentrations on dispersed bovine parathyroid cells.

We investigated the effects of the basic peptides polyarginine, protamine, and polylysine on dispersed bovine parathyroid cells. All three peptides produced a dose-dependent inhibition of dopamine-stimulated cAMP accumulation, with half-maximal inhibition at 4 x 10(-8), 1.5 x 10(-7), 3 x 10(-7), and 2 x 10(-6) M, respectively, for polyarginine, protamine, and two preparations of polylysine of molecular weights 10,200 and 3800. The inhibition of cAMP accumulation was reversible and was blocked by preincubating the cells overnight with 0.5 micrograms/ml of pertussis toxin. The same peptides also inhibited PTH release at similar concentrations, markedly stimulated the accumulation of inositol phosphates at two- to threefold higher concentrations, and produced transient increases in the cytosolic Ca2+ concentration (Cai) in fura-2-loaded parathyroid cells. The polylysine-evoked spike in Cai persisted despite the removal of extracellular Ca2+, indicating that it arose from intracellular Ca2+ stores. Exposure of the cells to elevated extracellular magnesium (Mg2+) concentrations elicited a similar spike in Cai but blocked the Cai transient in response to subsequent addition of polylysine, or vice versa. Thus, Mg2+ and polylysine mobilize Ca2+ from the same intracellular store(s). These results indicate that highly basic peptides closely mimic the effects of polyvalent cations on parathyroid function, suggesting that both agents may regulate parathyroid function via similar biochemical pathways.     

Mechanisms underlying the stimulation of PTH release by GppNHp in permeabilized bovine parathyroid cells

We examined changes in cAMP and inositol phosphate metabolism to assess the contribution of the guanine nucleotide regulatory (G) protein(s) regulating adenylate cyclase and phospholipase C in mediating the stimulatory effects of GppNHp on PTH release from permeabilized bovine parathyroid cells. To examine the role of Gs, the G protein stimulating adenylate cyclase, and cAMP on PTH release, permeabilized cells were incubated with either GppNHp or isoproterenol, and the effects of these agents on PTH release and cellular cAMP content were determined by RIA. To study the effects of GppNHp on inositol phosphate accumulation, permeabilized cells prelabeled with [3H]inositol were exposed to GppNHp, and inositol phosphates were measured using ion-exchange chromatography. These studies revealed that isoproterenol produced a dose-dependent increment in cAMP content in permeabilized cells with no significant effect on PTH release. Conversely, GppNHp rapidly and markedly elevated PTH release with a smaller and delayed rise in cAMP content. GppNHp- also promoted a dose-dependent increase in inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) accumulation, suggesting activation of phosphoinositide hydrolysis. Addition of dioctanoylglycerol, however, a synthetic diacylglycerol (DG) that activates protein kinase C, produced a much smaller increment in PTH release than GppNHp. Moreover, reducing the free calcium concentration to less than 10(-9) M by adding 10 mM EGTA to the permeabilization medium dissociated the effects of GppNHp and DG on secretion, increasing GppNHp-stimulated PTH release while reducing PTH secretion evoked by DG.(ABSTRACT TRUNCATED AT 250 WORDS)     

The diltiazem analog TA-3090 mimics the actions of high extracellular Ca2+ on parathyroid function in dispersed bovine parathyroid cells

We previously showed that the calcium channel blocker diltiazem raises cytosolic Ca2+ and inhibits PTH release in bovine parathyroid cells. To investigate further possible mechanisms underlying these effects, we examined the effects of the more potent diltiazem analog TA-3090, which is a Ca2+ channel antagonist in vascular smooth muscle, on several aspects of the function of dispersed bovine parathyroid cells. Like diltiazem, TA-3090 (10(-6)-10(-4] produced a dose-dependent inhibition of immunoreactive PTH release at 0.5 mM Ca2+ and raised the cytosolic Ca2+ concentration by 25-50% in fura-2-loaded parathyroid cells in the presence but not in the absence of extracellular Ca2+, suggesting that it activated rather than inhibited Ca2+ channels. To determine whether this compound affects other aspects of parathyroid function, we examined its effects on the inhibition of cAMP accumulation by Ca2+, a process we recently found to involve inhibition of cAMP generation by Gi through a receptorlike mechanism, which is independent of changes in cytosolic Ca2+. TA-3090 (10(-4) M) inhibited dopamine-stimulated cAMP accumulation by up to 75% (from 663 to 166 fmol per 10(5) cells), with a higher apparent potency at greater extracellular Ca2+ concentrations. Moreover, the addition of 10(-4) M TA-3090 potentiated the inhibitory effects of both Ca2+ and Mg2+, decreasing the concentration of the divalent cation necessary to produce half-maximal inhibition of cAMP accumulation by about twofold. In the absence of extracellular Ca2+, however, TA-3090 had no effect on the stimulation of cAMP by dopamine or on the inhibition of dopamine-stimulated cAMP accumulation by PGF2 alpha, which also regulates cAMP via Gi.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphoinositide hydrolysis and insulin release from isolated perifused rat islets. Studies with glucose

The ability of glucose to promote the hydrolysis of prelabeled [2-3H]inositol-containing phosphoinositides (PI) was assessed by measuring the efflux of 3H in response to glucose and the accumulation of labeled inositol phosphates. The inclusion of nonradioactive inositol (1 mM) in the perifusion medium dramatically improved our ability to monitor glucose-induced increases in 3H efflux. Efflux studies with this method revealed the following. 1) 3H efflux is significantly greater at 7 than at 2.75 mM glucose, and this parallels a small but significant increase in insulin secretion. 2) D-manno-Heptulose reduces 3H efflux with 7 mM glucose to a level approximating that seen in the presence of 2.75 mM glucose and has no effect on 3H efflux with 2.75 mM glucose. 3) In the presence of 20 mM glucose plus 1 mM inositol, 3H efflux is rapid and biphasic, a response that parallels the timing and amplitude of the biphasic pattern of insulin secretion. Direct measurements of labeled inositol and inositol phosphate levels in islets revealed the following. 4) After 50 min of perifusion with 2.75 or 7 mM glucose, labeled inositol phosphates were significantly greater with 7 mM glucose. 5) In response to 20 mM glucose alone, islet levels of free inositol, inositol monophosphate (IP1), and inositol bisphosphate (IP2) increased. 6) In response to 20 mM glucose plus 1 mM cold inositol, islet levels of free inositol increased, whereas islet levels of IP1, IP2, and inositol trisphosphate (IP3) were reduced compared with values obtained with 20 mM glucose alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipase C activation by endothelin-1 and noradrenaline in isolated penile erectile tissue from rabbit.

The effects of endothelin-1 and noradrenaline on phospholipase C activity in the rabbit isolated corpus cavernosum were investigated by measuring the accumulation of inositol phosphates. Both endothelin-1 and noradrenaline caused a time- and concentration-dependent increase in the accumulation of 3H-inositol phosphates in preparations prelabelled with 3H-myo-inositol. The reaction was slow in onset with no significant accumulation of 3H-inositol phosphates, including inositol trisphosphate, demonstrable during the first 15 minutes. At 60 minutes, the mean increases in 3H-inositol inositol phosphates induced by 3 x 10(-7) M endothelin-1 and 10(-3) M noradrenaline amounted to 341 and 530% of time-matched controls, respectively. However, when given at concentrations having the same contractile amplitude on rabbit corpus cavernosum, there was no difference in the amounts of 3H-inositol phosphates generated by endothelin-1 and noradrenaline. Prazosin (10(-6) M) significantly inhibited the stimulatory effect of noradrenaline on phosphoinositide hydrolysis. Pretreatment with 10(-6) M nimodipine did not reduce the increases in 3H-inositol phosphates induced by 3 x 10(-7) M endothelin-1 and 10(-3)M noradrenaline. Also in Ca(2+)-free medium, both agonists had significant stimulatory effects on phosphoinositide turnover, although under this condition, the responses were greatly reduced. The results suggest that exogenous endothelin-1 and noradrenaline activate phospholipase C in corpus cavernosum, and that this mechanism is partly independent of extracellular Ca2+. Considering the slow onset of action, phospholipase C activation is probably not directly involved in rapid contractile events, but may be of importance in the long-term regulation of penile smooth muscle tone.     

Stimulation of inositol phosphate formation in ROS 17/2.8 cell membranes by guanine nucleotide, calcium, and parathyroid hormone

In addition to stimulation of cyclic AMP, parathyroid hormone (PTH) may influence cellular events by utilizing other pathways of hormone action, such as the generation of inositol phosphates (IPs). We sought to examine this potential action of PTH by assessing the formation of inositol phosphates in PTH-sensitive ROS 17/2.8 cells. The polyphosphoinositides were labeled by growing the cells with [3H]inositol following which cell homogenates were prepared. The nonhydrolyzable guanine nucleotide, GTP gamma S, and calcium ion, alone and together, stimulated all three IPs, IP1, IP2, and IP3. IP1 formation was linear over 30 minutes but IP2 and IP3 accumulated more rapidly peaking by 5 minutes for all agonist conditions. The proportion of total P as IP3 was enhanced when the cells were grown with retinoic acid (1 microM) or when the assay was conducted at pH 4.5. In addition, the lower pH was associated with much more enzyme activity. PTH agonists, bPTH-(1-84) and bPTH-(1-34), both caused a small but significant stimulation of IP3 formation. When bPTH-(1-84), and the analog bPTH-(3-34)amide, that inhibits PTH-mediated adenylate cyclase activity were present together, there was additive stimulation of IP3 formation compared with that with either agent alone. The results demonstrate that inositol phosphate formation can be stimulated directly in a membrane preparation of ROS cells by GTP gamma S, calcium ion, and PTH and that the enzyme mediating this activity, phospholipase C, is regulated by a guanine nucleotide binding protein.     

Phorbol ester (TPA) reduces prostaglandin E2-stimulated cAMP production by desensitization of prostaglandin E2 receptors in a clonal osteoblast-like cell line,MOB 3-4

Summary A clonal osteoblast-like cell line, MOB 3-4, increased cAMP production in response to prostaglandin E₂ (PGE₂) (5–500 ng/ml). The purpose of this study was to show the effects of tumor-promoting phorbol ester (e.g., 12-O-tetradecanoylphorbol 13-acetate, TPA) on basal and PGE₂-stimulated cAMP production and the affinity of PGE₂ receptors in the cells. Pretreatment with TPA (1 nM–10 μM) for 30 minutes increased basal cAMP production, whereas it markedly reduced the PGE₂-stimulated cAMP production in the presence of 0.1 mM isobuthylmethyl xanthine. Both the TPA increase and reduction were dose- and time-dependent. However, TPA exerted no effect on forskolinor cholera toxin-stimulated cAMP production. Copretreatment with TPA and H-7, an inhibitor of protein kinase C (PKC), prevented the TPA-induced increase in basal cAMP production, whereas it did not prevent the reduction of the PGE₂-stimulated cAMP production. On the other hand, TPA (0.1–10 μM) decreased³H-PGE₂ binding in a dose- and time-dependent manner. Scatchard analysis revealed that TPA decreased the apparent affinity of PGE₂ receptors without effect on their apparent number. In addition, 1-oleoyl-2-acetylglycerol (12.6 μM), a synthetic diacylglycerol analog, did not mimic the TPA action on³H-PGE₂ binding. Thus, TPA at relatively high concentrations appeared to increase basal cAMP production by a PKC-mediated mechanism, and it appeared to directly act on PGE₂ receptors to decrease their apparent affinity and thereby reduce the PGE₂-stimulated cAMP production in the clonal osteoblast-like MOB 3-4 cell line.     

Actions of rat growth hormone-releasing factor and norepinephrine on cytosolic free calcium and inositol trisphosphate in rat C-cells

Rat growth hormone-releasing factor (rGRF) and norepinephrine (NE) stimulate secretion of calcitonin (CT) and neurotensin (NT) from cultured C-cells. The mechanism by which these agents cause secretion has not been well studied. We have examined the actions of the CT and NT secretagogues rGRF and NE on cytosolic free calcium concentrations ([Ca2+]i) in the rat C-cell line rMTC 44-2. Because inositol trisphosphate (IP3) has been shown to cause release of intracellular calcium stores in several cell types, we have also examined the effects of rat GRF, NE, and increases in extracellular calcium on IP3 accumulation in rMTC 44-2 cells. Stimulation by 10(-6) M rGRF caused a biphasic response in [Ca2+]i consisting of a rapid spike to 136 +/- 4% (mean +/- SE) of basal [Ca2+]i. This increase in [Ca2+]i decayed to base line and then gradually increased to 173 +/- 13% of basal [Ca2+]i. Stimulation by 10(-6) M NE gave a similar biphasic increase in [Ca2+]i. The increases in [Ca2+]i induced by both rGRF and NE were inhibited by pretreatment with EGTA or verapamil. rGRF, NE, and increasing concentrations of extracellular calcium, which all caused rapid increases in [Ca2+]i, failed to increase IP3 accumulation in rMTC 44-2 cells. These results suggest that rGRF- and NE-induced secretion in C-cells are mediated by changes in [Ca2+]i. These increases in [Ca2+]i appear to be generated by extracellular calcium influx rather than by release of intracellular calcium stores.     

G protein-dependent activation of a phosphoinositide-specific phospholipase C in UMR-106 osteosarcoma cell membranes

Recent evidence suggests that guanyl nucleotide binding (G) proteins are involved in receptor-mediated bone resorption and in osteoblastic function, but the nature of the G protein coupled to effectors that are involved in these skeletal effects is unknown. The purposes of this study were to determine (1) whether a G protein mediates activation of phosphoinositide-specific phospholipase C in UMR-106 rat osteosarcoma cells, and (2) whether parathyroid hormone (PTH) and a PTH-like protein (PLP) associated with humoral hypercalcemia of malignancy promote GTP-dependent PIP2 hydrolysis. Addition of GTP (10(-4) M) or guanosine 5'-0-(3-thiotriphosphate, GTP gamma S, 10(-5) M) to membranes prepared from UMR-106 cells labeled with [3H]myo-inositol increased both [3H]inositol trisphosphate (IP3) and [3H]inositol bisphosphate (IP2) formation. The increases in [3H]IP2 and [3H]IP3 produced by GTP were 8.6- and 4.3-fold, respectively. GTP gamma S produced a 17.6- and 11.9-fold increase in [3H]IP2 and [3H]IP3, respectively. The stimulatory effects of GTP and GTP gamma S were dose dependent (GTP ED50 = 3.9 x 10(-6) M; GTP gamma S ED50 = 2.5 x 10(-7) M) and progressive over 10 minutes and required the presence of Mg2+.GTP (10(-4) M) and GTP gamma S (10(-5) M) decreased membrane [3H]phosphoinositides concomitantly with increased [3H]IP2 and [3H]IP3. The GDP analog guanosine 5'-O-(2-thiodiphosphate, GDP beta S) alone did not alter [3H]IP2 or [3H]IP3 production but at 10(-4) M blocks the stimulatory effects of GTP and GTP gamma S. NaF (3 x 10(-2)M) produced a 2.8- and 2.0-fold stimulation of [3H]IP2 and [3H]IP3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of guanine nucleotides and parathyroid hormone on inositol 1,4,5-trisphosphate metabolism in canine renal cortical tubular cell membranes

Parathyroid hormone (PTH) and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma S) increase levels of the second messenger inositol 1,4,5-triphosphate (IP3) and other inositol phosphates (IP) in several membrane preparations of PTH-responsive cells. We present evidence here indicating that in a membrane preparation of canine renal cortical tubular cells bPTH-(1-84), bPTH-(1-34), [N-Leu8,18Tyr34]bPTH-(3-34)NH2, and the human PTH related peptide fragment hPTHrP-(1-34)NH2 all increase levels of inositol phosphate (IP) but [Tyr34]-bPTH-(7-34)NH2 and hPTHrP-(7-34)NH2 have no significant effects on IP accumulation. Increases in IPs are generally attributed to increased formation of IPs and appear to be mediated by a G protein. However, increased levels of IPs may also result from inhibition of the phosphatases are responsible for their metabolism. We investigated the effect of PTH and GTP-gamma S on the metabolism of IP3 in canine renal cortical tubular membranes. These membranes rapidly metabolize [3H]IP3 (47% at 15 s). Decreases in [3H]IP3 at all time points are accounted for quantitatively by increases in the sum of its breakdown products: [3H]IP2, [3H]IP1, and [3H]inositol. After 5 minutes of exposure to membranes, the vast majority of [3H]IP3 (84%) is converted to its terminal metabolite, [3H]inositol. GTP-gamma S (100 microM) inhibits the amount of [3H]IP3 metabolized in 15 s by 70% and reduces the amount of [3H]inositol ultimately formed in 5 minutes by 64%. ATP-gamma S, ATP, and 2,3-bisphosphoglycerate (100 microM) also inhibit [3H]IP3 hydrolysis in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purinergic regulation of cytosolic calcium and phosphoinositide metabolism in rat osteoblast-like osteosarcoma cells.

We have shown that ATP increases cytosolic Ca2+ in UMR-106 cells through P2-purinergic receptor stimulation (Calcif Tissue Int 45:251-254). This response was further characterized using cells loaded with indo-1/AM or prelabeled with [3H]inositol. ATP elicited a rapid transient increase in Ca2+ from 148 to 540 nM, followed by a biphasic decline (first rapid and then slower) to basal within 1 minute and then a late slow rise to 200 nM by 4 minutes. ADP also elicited a rapid transient increase, but this was followed by a second smaller transient and a later, slow increase above basal Ca2+. These transient increases in Ca2+ induced by ATP and ADP were dose dependent, detected at 10(-6)M ATP and 10(-7)M ADP, and saturated at 10(-4)M with both nucleotides. The maximum increase in Ca2+ was 20% greater with ATP than ADP. EGTA chelation of extracellular Ca2+ abolished the biphasicity of the ATP-induced Ca2+ transient, the second ADP-induced transient, and all late slower increases in Ca2+. Desmethoxyverapamil pretreatment attenuated the biphasicity of the ATP-induced transient and the second peak elicited by ADP. Elevated extracellular Ca2+ (5 mM) prevented the return to the basal level that normally follows the ATP-induced Ca2+ transient and amplified the sustained increase in Ca2+ but had little effect on the response to ADP. IP3 and IP4 increased rapidly after addition of ATP, with I(1,4,5)P3 increasing before I(1,3,4)P3. These data indicate that P2-purinergic stimulation of UMR-106 cells causes three consecutive responses in cytosolic Ca2+: (1) a transient increase due to IP3-mediated mobilization of intracellular Ca2+; (2) a transient increase due in part to influx, probably associated with a Ca2+ channel; and (3) a later sustained increase that requires extracellular calcium.     

Effects of extracellular calcium and magnesium on cytosolic calcium concentration in Fura-2-loaded bovine parathyroid cells

A newly developed calcium-sensitive dye, Fura-2, was employed in dispersed bovine parathyroid cells to study the effects of extracellular calcium and magnesium on cytosolic calcium concentration and parathyroid hormone (PTH) release. In comparison with control cells, Fura-2-loaded parathyroid cells showed the same maximal rate of PTH release, set-point for extracellular Ca++ (the calcium concentration producing half of the maximal inhibition of PTH release), and maximal inhibition of PTH release (71.6%) by high extracellular Ca++. At an extracellular Mg++ concentration of 0.5 mM, raising extracellular Ca++ in a stepwise fashion from 0.5 mM to 2.0 mM produced a dose-dependent, statistically significant (p less than 0.01) increase in cytosolic Ca++ from 198 +/- 24 nM (0.5 mM Ca++) to 411 +/- 21 nM (2.0 mM Ca++) which closely paralleled the concomitant decrease in PTH release. An elevation of extracellular Mg++ from 0.5 mM to 5 mM, at an extracellular Ca++ of 0.5 mM, resulted in a transient spike of cytosolic Ca++ which lasted for approximately 30 seconds, followed by a small but stable increase in the cytosolic Ca++ concentration (174 +/- 7 nM vs. 237 +/- 10 nM, n = 4, p less than 0.01). Prior removal of extracellular calcium by addition of an excess of EGTA did not abolish the transient spike induced by high extracellular magnesium concentrations in Fura-2-loaded cells, suggesting that this rapid increase in cytosolic Ca++ arises, at least in part, from intracellular stores of Ca++. This is supported by the observation that pretreating cells with ionomycin resulted in disappearance of the magnesium-induced spike.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Inhibition of Lysophosphatidate Signaling by Volatile Anesthetics

Background: Volatile anesthetics have been found to interfere with the functioning of several G protein-coupled receptors, effects that may be relevant to the mechanism of anesthetic action. Lysophosphatidate (1-acyl-2-sn-glycero-3-phosphate; LP) is the simplest natural phospholipid. It has pronounced biological effects and signals through a specific G protein-coupled receptor. Because of its lipophilicity, the LP receptor is a feasible site of anesthetic interaction. Therefore, the authors investigated the effects of halothane and isoflurane on LP signaling using Xenopus oocytes.

Methods: Mature oocytes were harvested from Xenopus frogs, isolated, and defolliculated manually. Lysophosphatidate receptors are endogenously present in these cells. Angiotensin receptors were expressed recombinantly to study anesthetic effects on intracellular signaling. Oocytes were studied individually with a two-electrode voltage clamp at room temperature. Integrated Ca2+ -activated Cl sup - currents (ICl(Ca)) were used to evaluate the effects of anesthetics on changes in intracellular Ca2+ concentration in response to receptor agonists (10 sup -7 M LP or 10 sup -7 M angiotensin II) or intracellular inositoltrisphosphate (IP3) injection.

Results: Halothane depressed LP signaling in a concentration-dependent manner, with half-maximal inhibition at 0.23 mM and virtually complete inhibition at 0.34 mM. Responses could be recovered after an anesthetic-free wash. Oocyte injection with heparin, an IP3 receptor antagonist, completely blocked LP and angiotensin signaling, indicating similar IP3 -dependent pathways. However, ICl(Ca) induced by angiotensin receptor activation or intracellular IP3 injection were not inhibited by halothane. Isoflurane, at comparable concentrations, did not depress LP responses in oocytes significantly.     

Regulation by calcium and 1,25-(OH)2D3 of cell proliferation and function of bovine parathyroid cells in culture

The effects of high calcium and 1,25-(OH)2D3 on parathyroid cell growth, PTH secretion, and steady-state levels of pre-proPTH mRNA in proliferating bovine parathyroid cells were examined. Cells were established in primary tissue culture and then tested in passages 2 and 5. Cell proliferation was suppressed by 10(-9)-10(-7) M 1,25-(OH)2D3 but not by high calcium (2.5 mM). Cells at passages 2 and 5 were grown to subconfluence and then exposed for 72 h to 2.5 mM calcium or 10(-7) M 1,25-(OH)2D3. Pre-proPTH mRNA was decreased to approximately 50% of control by 2.5 mM calcium compared with 0.3 and 1.0 mM calcium. PTH secretion, as tested by low calcium stimulation for 1 h at the end of 72 h incubation, was inhibited by 50% in cells that had been exposed to high calcium compared with control. Incubation with 10(-7) M 1,25-(OH)2D3 caused a decrease in the levels of pre-proPTH mRNA and PTH release to 50% of control at 72 h. These results suggest that cultured bovine parathyroid cells, at least in early passages, have responses to high calcium and 1,25-(OH)2D3 similar to those in primary nonproliferating cultures studied earlier and that 1,25-(OH)2D3 inhibits the proliferation of parathyroid cells in a dose-responsive fashion.     

Ketamine suppresses norepinephrine-induced inositol 1,4,5-trisphosphate formation via pathways involving protein kinase C

Inositol 1,4,5-trisphosphate (IP(3)) is not only involved in the physiologic regulation of excitation-contraction coupling, but could also play a role in cardiac pathophysiology. We investigated the mechanism of ketamine modulation of norepinephrine (NE)-induced IP(3) formation in neonatal rat cardiomyocytes. Ketamine 1 and 10 microM significantly decreased the IP(3) response to 1 microM NE by 27% and 43%, respectively. One micromolar TMB-8 (an intracellular calcium antagonist) produced 42% more decreases in IP(3) production than produced by ketamine alone. One hundred micromolar anthranilic acid (a phospholipase A(2) inhibitor) significantly decreased NE (1 microM)-induced IP(3) formation, and the inhibition was further enhanced by ketamine. Ten micromolar U 73122 (a phospholipase C inhibitor) did not significantly affect NE-induced IP(3) in the presence or absence of ketamine. One micromolar ketamine significantly inhibited staurosporine (a nonselective protein kinase C antagonist)-, bisindolylmaleimide (a selective protein kinase C antagonist)-, and wortmannin (a phosphatidylinositide 3-kinase antagonist)-stimulated IP(3) formation. In conclusion, ketamine suppresses NE-induced IP(3) production, and the inhibition is caused through pathways including protein kinase C and a decrease in intracellular Ca(2+) concentrations. IMPLICATIONS: Ketamine inhibits norepinephrine-induced inositol 1,4,5-triphosphate formation in a dose-dependent manner via pathways that involve protein kinase C and a decrease in intracellular Ca(2+) concentrations.     

Synergistic inhibition of lysophosphatidic acid signaling by charged and uncharged local anesthetics.

We investigated the mechanism of benzocaine (permanently uncharged) and QX314 (permanently charged) inhibition of lysophosphatidic acid (LPA) signaling. To determine their site of action, we studied effects of these drugs, alone and in combination, on LPA-induced Ca2+-dependent Cl currents (I(Cl(Ca))) in Xenopus oocytes. After 10 min exposure to benzocaine, QX314 (10(-6)-10(-2) M), or both, we measured effects on I(Cl(Ca)) induced by LPA (with and without protein kinase [PKC] activation/inhibition) and on I(Cl(Ca)) induced by the intracellular injection of IP3 and GTPgammaS. LPA application to oocytes resulted in I(Cl(Ca)) (50% effective concentration approximately 10(-8) M). Both anesthetics inhibited LPA signaling concentration-dependently (50% inhibitory concentration [IC50] benzocaine 0.9 mM, QX314 0.66 mM). The combination acted synergistically (IC50 benzocaine 0.097 mM/QX314 0.048 mM). Intracellular signaling pathways were not affected. This study shows that benzocaine and QX314 inhibit LPA signaling and act synergistically, which is most easily explained by the existence of two different binding sites. Lack of inhibition of IP3 or GTPgammaS-induced I(Cl(Ca)) identifies the receptor as a target. Activation of PKC can be excluded as a potential mechanism. IMPLICATIONS: Lysophosphatidic acid may play a role in wound healing, and its signaling is inhibited by local anesthetics. We identified the membrane receptor as the local anesthetic site of action and showed that charged (QX314) and uncharged (benzocaine) local anesthetics inhibit lysophosphatidic acid signaling synergistically, which can be explained by the presence of different binding sites.     

Parathyroid hormone stimulates formation of inositol phosphates in a membrane preparation of canine renal cortical tubular cells

Recent studies have shown that, in addition to its well-known action to stimulate adenylate cyclase activity, parathyroid hormone (PTH) may stimulate the inositol phosphate second messenger system in its target tissues, bone and kidney. We have developed a membrane preparation of canine renal cortex to test this hypothesis. We also have examined the potential role of guanine nucleotides on the formation of inositol phosphates (IPs) in this tissue. Collagenase-dispersed tubules were labeled with [3H]inositol, and membranes containing labeled phospholipase C (PLC) substrates ([3H]phosphatidyl inositol, [3H]phosphatidylinositol monophosphate, and [3H]phosphatidylinositol bisphosphate) were prepared. bPTH-(1-34) (100 nM) rapidly increased levels of all measured [3H]IPs (IP1, IP2, and IP3) 1.6-1.7-fold within the first 30 s of stimulation. The half-maximal concentration for the response to bPTH-(1-34) was approximately 8 nM. GTP gamma S (100 microM), a nonhydrolyzable analog of GTP, also increased levels of the three [3H]IPs (1.8 to 2.8-fold). The half-maximal concentration for the response to GTP gamma S was approximately 30 microM. In the presence of GTP gamma S, bPTH-(1-34) increased levels of IPs by up to 2.7 times more than GTP gamma S alone. The results indicate that bPTH-(1-34) can stimulate the formation of inositol phosphates in the kidney and suggest that PTH may activate a receptor coupled to this effect through a guanine nucleotide regulatory protein.     

Inhibitory Effects of Propofol on Intracellular Signaling by Endothelin-1 in Aortic Smooth Muscle Cells

Background: Blood pressure decreases when propofol is administered. However, the exact mechanism underlying the vascular effects of propofol has not yet been elucidated. Endothelin produced by vascular endothelial cells is a potent vasoactive peptide that elicits prolonged contraction of vascular smooth muscle cells. The effects of propofol on endothelin-1-induced intracellular signaling in an aortic smooth muscle cell line, A10 cells, were examined.

Methods: Cultured A10 cells were pretreated with propofol for 20 min and then stimulated with endothelin-1. The effect of propofol on the endothelin-1-induced Ca2+ influx into A10 cells was evaluated by measuring intracellular45 Ca2+. The effects of propofol on the endothelin-1-induced activation of phosphatidylinositol-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D were evaluated by measuring the formation of inositol phosphates and choline, respectively. The effect of propofol on endothelin-1 binding to its receptor was determined by an [sup 125 I] endothelin-1-binding assay.

Results: Propofol inhibited the the endothelin-1-induced Ca2+ influx, but this was significant only at supuraclinical concentrations. The endothelin-1-stimulated formation of inositol phosphates was significantly suppressed by propofol. However, propofol had no effect on the formation of inositol phosphates induced by NaF, an activator of heterotrimeric guanosine triphosphate (GTP)-binding proteins. Propofol inhibited the endothelin-1-induced formation of choline. Propofol had no effect on the binding of endothelin-1 to its receptor.