Cyclic AMP production in rat calvaria in vitro: interaction prostaglandins with parathyroid hormone.

We studied the cAMP response of cultured rat calvaria to parathyroid hormone (PTH) and prostaglandins (PG) in order to test the hypothesis that PTH action is mediated by PG. PTH and PGE1 each caused an 8-fold increase in tissue cAMP during 15 min incubation. There was an additive response to the combination of the two agonists at maximally effective individual concentrations. Similar results were obtained when adenylate cyclase activity in bone homogenates were measured. Calvaria incubated for 60 min with PGE1 were desensitized to further stimulation by PGE1, but responded normally to PTH. Indomethacin, aspirin and phenylbutazone, 10(-5)M, had no effect on the cAMP response to PTH. At millimolar concentration these agents did block the hormone response; however, the response to exogenous PGE1 and the fluoride-sensitive adenylate cyclase were also inhibited. Thus, it appears that the effects of high concentrations of the anti-inflammatory drugs on cAMP formation are non-specific. Our results suggest that receptors in bone for PTH and prostaglandins are separate and independent, and that the cAMP response to PTH is not mediated by prostaglandins.     

Metabolism of parathyroid hormone by fetal rat calvaria.

These studies examine the metabolism of highly purified bovine parathyroid hormone [bPTH-(1--84)] by fetal rat calvaria. Enzymatically dispersed bone cells and intact (minced) calvaria were incubated with bPTH-(1--84) and the incubation medium was analyzed for degradation of PTH by polyacrylamide gel electrophoresis. Eluates of gel slices were assayed for immunoreactive PTH (iPTH) in carboxy- and amino-terminal RIAs. Both bone preparations metabolized bPTH-(1--84). The intact hormone progessively decreased with time and carboxy-terminal iPTH fragments were evident by 5 min of incubation. In the isolated cell preparations, intact hormone was completely degraded at submaximal doses of PTH (5 X 10(-9) M), as assessed by cAMP production. Degradation was incomplete in intact calvarial preparations at all doses studied. Intact calvaria were less sensitive to PTH with regard to cAMP production. No amino-terminal fragments were detected in the medium with either cell preparation. Oxidized (biologically inactive) bPTH-(1--84) was not metabolized in these systems. These findings contrast with studies in liver and kidney preparations, where oxidized bPTH has been shown to be degraded. These findings contrast with studies in liver and kidney preparations, where oxidized bPTH has been shown to be degraded. These data suggest that biological activity may be necessary for the metabolism of intact bPTH-(1--84) by bone cells and that skeletal tissue may contribute to the immunoheterogeneity of circulating PTH in the rat.     

Mechanism of regulation of prostaglandin production by parathyroid hormone, interleukin-1, and cortisol in cultured mouse parietal bones

Bovine PTH-(1-34) (PTH), human recombinant interleukin-1 alpha (IL-1), and cortisol were tested for their effects on bone resorption, prostaglandin (PG) production, and PG endoperoxide synthase (PGH synthase or cyclooxygenase) mRNA levels in cultured mouse parietal bones. Cultures were treated with PTH and IL-1 in the presence and absence of cortisol and arachidonic acid (AA). We found that both PTH and IL-1 stimulated the release of PGE2 and 6-keto-PGF1 alpha (the stable metabolite of PGI2). Stimulation of each metabolite by IL-1 at 0.6-60 pM was 2- to 118-fold, and that by PTH at 24 pM to 24 nM was 3- to 53-fold. Thus, IL-1 was 40-fold more potent than PTH in stimulating PG release. Moreover, IL-1 showed 2- to 3-fold greater efficacy than PTH in stimulating PGE2 release. However, IL-1 was only 4-fold more potent and no more effective than PTH in stimulating 45Ca release. IL-1 (60 pM) and PTH (2.4 nM) stimulation of PGE2 production showed a similar time course, with a lag phase of 0.75-1.5 h. Cortisol (1-100 nM) reduced basal PGE2 production and calcium release. The absolute amounts of PG produced in response to PTH and IL-1 were reduced in the presence of cortisol, but in the presence of AA the relative increases were still from 2.5- to 26-fold compared with levels in cultures treated with cortisol alone. Cortisol reduced the stimulation of 45Ca release by IL-1, but not by PTH. AA (10(-5) M) amplified PG production in response to PTH and IL-1, but not 45Ca release. In bones labeled with [3H]AA, IL-1 and PTH increased [3H]PGE2 and [3H]6-keto-PGF1 alpha release, as measured by HPLC and TLC. IL-1 slightly increased [3H]AA release, but PTH did not. Cortisol decreased [3H]AA release. To test for an effect on PG production at the level of PGH synthase, mRNA levels were measured. mRNA was increased by both PTH and IL-1 to a similar extent despite the greater effect of IL-1 on PGE2 production. Cortisol did not change PGH synthase mRNA levels and did not block the stimulation by PTH or IL-1. We conclude that IL-1 is a more potent stimulator of PG production and bone resorption than PTH. Stimulation of PG production by both PTH and IL-1 is mediated at least in part by increasing PGH synthase, but IL-1 may have an additional effect on AA release.     

Effects of gonadotrophin-releasing hormone on testosterone secretion by fetal and neonatal rat testes in vitro

Testosterone production by fetal (20.5 days) and neonatal (1-day-old) rat testes was measured to study the possible direct effect of gonadotrophin-releasing hormone (GnRH) on steroidogenesis in early development. Single gonads were incubated in the presence and absence of GnRH (10(-10) to 10(-6) M) or agonistic analogues (10(-11) to 10(-7) M). For comparison, some testes were incubated with ovine luteinizing hormone (0.001-0.01 microgram oLH1 NIH-LH-S20/ml of medium TC199. No clear evidence of stimulation by an agonist (10(-7) M) was seen with fetal testes, but the lowest concentration (10(-11) M) gave results suggestive of an inhibitory action. Similar experiments with neonatal testes showed stimulation. With the highest concentration of GnRH or agonist the amounts of testosterone produced were about 2-3-fold greater than from control testes. Greater quantities of testosterone were found with testes exposed to LH. Hourly sampling in one experiment showed that significant stimulation of testosterone secretion had occurred within the first hour with GnRH (10(-6) M). It was concluded that fetal-neonatal rat Leydig cells are responsive to GnRH.     

Effects of prostaglandin H2, prostaglandin E2, and arachidonic acid on parathyroid hormone and antidiuretic hormone activation of rat kidney adenylate cyclase

These experiments examine interactions of arachidonic acid; the substrate for prostaglandin cyclooxygenase, prostaglandin (PG)H₂, a key endoperoxide intermediate in prostaglandin synthesis; and prostaglandin (PG)E₂, an important prostaglandin produced within the kidney; with adenylate cyclase activity in renal cortex, outer medulla, and inner medulla. In addition, the effects of arachidonic acid, PGH₂, and PGE₂ on parathyroid hormone (PTH) activation of adenylate cyclase in cortex, and of antidiuretic hormone (ADH) activation of that enzyme in outer and inner medulla are examined. Arachidonic acid elicited a concentration-dependent inhibition of basal and PTH-stimulated adenylate cyclase activity in renal cortex. Concentration-dependent inhibition by arachidonic acid of basal and ADH-stimulated adenylate cyclase activity was observed in outer and inner medulla. PGH₂ inhibited basal activity in all three areas of the kidney. There was also inhibition by PGH₂ of medullary ADH and cortical PTH stimulation. PGE₂ stimulated adenylate cyclase in all three areas. PGE₂ had no effect upon PTH stimulation in cortex and was additive with ADH in outer and inner medulla. PGE₂ stimulation was inhibited by arachidonic acid, and this inhibition seemed competitive. Inhibition by both arachidonic acid and PGH₂ was not destructive. Experiments with [1-¹⁴C]arachidonic acid and indomethacin suggest that the inhibition by arachidonic acid was actually mediated by arachidonic acid and not a metabolite. Both PGH₂ and arachidonic acid inhibition was independent of phosphodiesterase. This activation by product, PGE₂, and inhibition by its precursors, arachidonic acid and PGH₂, provide a possible mechanism by which the prostaglandin system could modulate adenylate cyclase responsiveness to hormonal activation.     

The effects of parathyroid hormone on osteoblast-like cells from embryonic chick calvaria

A bone cell fraction was isolated from 16 day old embryonic chick calvaria using a sequential enzymatic digestion procedure. The fraction contained cells, of an osteoblast-like character, which responded to parathyroid hormone (PTH) and prostaglandin E2, but not to calcitonin, in terms of increased production of cyclic AMP. Primary cultures of cells maintained their responsiveness to PTH for at least 2 weeks after reaching confluence. Production of alkaline phosphatase by the bone cells was inhibited when 1,25(OH)2 vitamin D3 was added to cultures at concentrations of 10(-8)M or greater. When cells were cultured in the presence of PTH a biphasic effect was observed; alkaline phosphatase levels were stimulated at low concentrations of this hormone but were decreased at higher concentrations. The latter finding appears consistent with observations that PTH can in vivo exert either anabolic or catabolic effects on bone, depending upon the circulating level of hormone present.     

甲状旁腺素1-34对新生Wistar大鼠心肌成纤维细胞增殖的影响

目的观察人甲状旁腺素1-34(hPTH)对原代培养新生Wistar大鼠心肌成纤维细胞(CFs)增殖的影响,通过细胞内钙离子([Ca~(2 )]i)的变化,探讨诱导CFs增殖的发生机制。方法培养新生Wistar大鼠心肌成纤维细胞,根据加入不同浓度的hPTH(10~(-10)、10~(-9)、10~(-8)mol/L)分组、同时设维拉帕米(L型钙通道拈抗剂)组。四氮唑盐比色法(MTT)、~3H-TdR掺入法检测细胞增殖；激光共聚焦显微镜(LSCM)测定细胞内[Ca~(2 )]i。结果①CFs增殖活性组间比较,差异有统计学意义(F=9．20,P＜0．01)。随着hPTH的增加,CFs增殖活性呈明显递增性升高,10~(-10)、10~(-9)、10~(-8) mol/L组CFs增殖活性分别为(0．408±0．016)、(0．518±0．019)、(0．778±0．034),与对照组(0．365±0．013)比较,差异有统计学意义(P＜0．01)。②~3H-TdR的掺入量组间比较差异有统计学意义(F=8．82,P＜0．01)；~3H-TdR的掺入量也随着hPTH的升高而呈递增趋势,10~(-10)、10~(-9)、10~(-8) mol/L组分别为(36．79±2．03)、(39．13±1．98)、(45．51±2．64)Bq,与对照组(24．08±1．23)Bq比较,差异有统计学意义(P＜0．01)；维拉帕米组~3H-TdR掺入量(41．68±2．72) Bq,明显低于10~(-8) mol/L组(P＜0．01)。③CFs内[Ca~(2 )]j组间比较,差异有统计学意义(F=9．16,P＜0．01)；10~(-10)、10~(-9)、10~(-8) mol/L组CFs内[Ca~(2 )]i(38．28±4．40、63．78±5．15、78．39±6．87)也明显高于对照组(36．18±3．57)；维拉帕米组CFs内[Ca~(2 )]i为(52．14±4．69),明显低于单独应用hPTH 10~(-8) mol/L组。结论PTH能刺激CFs增殖,增殖活性与PTH浓度有关,L型钙通道开放引起的细胞外钙内流增加为其重要的机制之一。     

大鼠甲状旁腺素对体外培养心室肌细胞的促肥大作用

目的 观察不同浓度大鼠甲状旁腺素1-34（rat parathyroid hormone 1-34,rPTH1-34）对体外培养心室肌细胞的致肥大作用,观察细胞外信号调节激酶1/2（extracellular signal regulated kinases 1/2,ERK1/2）的变化。方法 体外培养新生大鼠心室肌细胞,以1×10-8～1×10-6 mol/L rPTH1-34分别孵育24 h,测定细胞直径、3H-亮氨酸(3H-Leu)掺入率、心房钠尿肽（atrial natriuretic peptide,ANP）mRNA、ERK1/2、磷酸化ERK1/2(p-ERK1/2)蛋白的表达。结果 与正常组相比,1×10-8 mol/L rPTH 1-34可使体外培养的心肌细胞直径、细胞蛋白合成速率轻度增加,ANP mRNA表达轻度升高,但无统计学意义;1×10-7,1×10-6 mol/L rPTH1-34 均可显著增加心肌细胞直径（P<0.01）,细胞蛋白合成速率（P<0.01）和ANP mRNA表达（P<0.01）。与正常组比较,PTH呈浓度依赖性地增加了ERK1/2和p-ERK1/2的表达（P<0.01）。结论 1×10-7,1×10-6 mol/L rPTH1-34能在体外诱导新生大鼠心室肌细胞发生肥大反应,且呈一定的浓度依赖性;PTH能呈浓度依赖性地刺激心肌ERK1/2过度表达。     

Stimulation of prostaglandin accumulation in the rat anterior pituitary gland by luteinizing hormone releasing hormone in vitro.

The addition of luteinizing hormone releasing hormone releasing hormone (LH-RH) to cultures of monolayers of rat anterior pituitary cells was shown to increase both the concentrations of prostaglandins E1 and E2 (PGE) in the cells and the release of LH over similar ranges of concentrations of LH-RH (10(-6) to 10(-10) mol/l). The peak concentration of PGE was observed after 2.5 h. The stimulation of the level of PGE in the cells by LH-RH was completely inhibited by two inhibitors of prostaglandin synthetase, which only partially inhibited the stimulation of LH release. Therefore the increased concentration of PGE was not obligatory for the effect of LH-RH on LH release. It was also shown that monobutyryl cyclic AMP stimulated the intracellular concentration of PGE and it is suggested that the stimulation of PGE levels may be mediated by increased levels of cyclic AMP in the cells after the addition of LH-RH.     

Stimulation of prostaglandin E and testosterone production in rat interstitial cells by a gonadotropin-releasing hormone agonist

The early direct effects of a GnRH agonist analog [D-Ala6]des-Gly10-GnRH N ethylamide (GnRHa) on rat testicular interstitial cells include increased production of prostaglandin E (PGE) and testosterone (T) at 3 h (ED50 values of 0.5 and 0.75 nM, respectively). On the other hand, LH action on testicular function, which is mediated by increased cAMP, involves an increase in T production at 30 min followed by increased PGE formation at 3 h. GnRHa at concentrations of 10(-12)-10(-8) M had no effect on basal or LH-stimulated cAMP production during a 4-h incubation test. The stimulatory effect of GnRHa on PGE, but not on T production, was abolished by the prostaglandin synthesis inhibitor indomethacin (1.5 microns). We conclude that cAMP does not play a role in mediating the direct testicular effects of GnRH on PGE and T production; that PGE is not involved in mediating GnRH-induced T production; and, finally, that increased PGE and T production might be involved in mediating the direct inhibitory and stimulatory testicular effects of GnRH and its agonist analogs.     

Follicle-stimulating hormone and somatomedin-C stimulate inhibin production by rat granulosa cells in vitro

The direct effect of somatomedin-C (Sm-C) and FSH on inhibin production by rat granulosa cells in vitro has been examined. FSH stimulated accumulation of inhibin in culture media in a dose-dependent manner with maximal stimulation (6-fold) being observed at a dose of 300 ng FSH/ml. Addition of Sm-C (30 ng/ml) either alone or in the presence of FSH (3-300 ng/ml) increased inhibin production (up to 5-fold). Sm-C alone was effective over the physiological dose range of 3-100 ng/ml. Concomitant addition of FSH (100 ng/ml) and Sm-C (3-100 ng/ml) resulted in a significant increase in inhibin production at all doses of Sm-C. The dose-dependent effects of FSH and Sm-C were also time dependent with a synergistic effect apparent after 48 h of culture. The Sm-C induced FSH inhibitory activity of granulosa cell culture media was confirmed as authentic inhibin by the demonstration of a dose-dependent neutralization of this activity by a monoclonal antibody raised against purified bovine inhibin. The data indicate a direct role for both FSH and Sm-C in ovarian inhibin production and provide additional evidence for an autocrine-paracrine role for Sm-C in granulosa cell differentiation.     

Effects of parathyroid hormone on cyclic-AMP concentrations of in vitro Necturus maculosus gastric antrum

The effects of bovine parathyroid hormone (bPTH1-84) on the stimulation of intracellular cyclic-AMP [cAMP] were investigated in an in vitro preparation of Necturus maculosus antral mucosa. When the antrum was exposed to 1, 5, 10, or 100 nM bPTH1-84, there was an approximately 2-fold nonlinear increase in tissue [cAMP] over basal values. The pretreatment of the antral mucosa with 1 mM isobutylmethylxanthine (IBMX, a phosphodiesterase inhibitor) increased with detectability of mucosal [cAMP]. The addition of 1, 5, 10, or 100 nM bPTH1-84 to tissues pretreated with IBMX resulted in an approximately 3.5-fold linear increase in mucosal [cAMP] over basal values. The time course of the generation of mucosal cAMP to 10 nM bPTH1-84 resulted in a small but significant transient increase at 2.5 min after the addition of bPTH1-84 but no change in the medium [cAMP]. In tissues pretreated with 1 mM IBMX the response to 10 nM bPTH1-84 was a large biphasic increase of [cAMP] at 2.5 min that progressively declined to near basal values by 15 min. There was also a significant sustained increase in the [cAMP] in the bathing medium at 2.5 min of tissues pretreated with IBMX followed by 10 nM bPTH1-84. These results suggest the presence of an adenylate cyclase that can be activated by a mammalian bPTH1-84 in elevating intracellular cAMP levels in the N. maculosus antral mucosa.     

Effects of galanin on hormone secretion from the in situ perfused rat pancreas and on glucose production in rat hepatocytes in vitro

Galanin is a novel peptide, widely distributed throughout the central and peripheral nervous system, including nerve endings surrounding the pancreatic islets. In dogs, galanin infusion has been reported to induce hyperglycemia along with a reduction of circulating insulin. In this work, we have studied the effect of galanin (a 200 ng bolus followed by constant infusion at a concentration of 16.8 ng/ml for 22-24 min) on insulin, glucagon, and somatostatin secretion in the perfused rat pancreas. In addition, we have investigated the effect of galanin (10 and 100 nM) on glycogenolysis and gluconeogenesis in isolated rat hepatocytes. In the rat pancreas, galanin infusion marked inhibited unstimulated insulin release as well as the insulin responses to glucose (11 mM), tolbutamide (100 mg/liter) and arginine (5 mM). Galanin failed to alter the glucagon and somatostatin responses to glucose, tolbutamide, and arginine. In isolated rat hepatocytes, galanin did not influence glycogenolysis or glucagon phosphorylase a activity. Gluconeogenesis and the hepatocyte concentration of fructose 2,6-bisphosphate were also unaffected by galanin. In conclusion: in the perfused rat pancreas, galanin inhibited insulin secretion without modifying glucagon and somatostatin output, thus pointing to a direct effect of galanin on the B cell; and in rat hepatocytes, galanin did not affect glycogenolysis or gluconeogenesis; hence, the reported hyperglycemia induced by exogenous galanin does not seem to be accounted for by a direct effect of this peptide on hepatic glucose production.     

Effects of glucocorticoids on secretion of luteinizing hormone and follicle-stimulating hormone by female rat pituitary cells in vitro

To determine if the divergent effects of glucocorticoids on the circulating levels of LH and FSH in female rats are exerted directly on the pituitary, adult female pituitary cells were treated either with no glucocorticoids or with 60 or 600 ng/ml cortisol or corticosterone during one or two 48-h incubations. During the second 48 h, some cells from each group were treated with GnRH (1.7 X 10(-12) - 4.6 X 10(-9) M). Concentrations of LH and FSH in media and cells were measured by RIA. Basal secretion of LH was inhibited 38-43% by different glucocorticoid treatment during the first 48 h and 21% by 600 ng/ml corticosterone during the second 48 h. In contrast, basal secretion of FSH was enhanced 22-64% during the first 48 h and 25-124% during the second 48 h. Secretion of LH in response to maximal stimulation with GnRH was unaffected by glucocorticoids, but maximal secretion of FSH was increased 68%. The responsiveness of the cells to GnRH, as determined from the slope of the GnRH dose-response curve for LH, was increased 43-50% by cortisol. The slope of the dose-response curve for FSH was unaffected, but the mean concentration of FSH as a function of the log dose of GnRH was increased 45-79%. Glucocorticoids had no effect on cell content of LH or total LH per dish, either under basal or maximal GnRH-stimulated conditions. Glucocorticoids increased basal cell content of FSH 41-82%, basal total FSH 35-93%, and maximal GnRH-stimulated total FSH 40-84%. These results suggest that the only negative effect of glucocorticoids on reproduction exerted at the level of the pituitary is a slight suppression of basal LH secretion, that glucocorticoids affect the pituitary directly by increasing FSH synthesis, and that the divergent effects of glucocorticoids on LH and FSH provide a novel model for differential regulation of the gonadotropins.     

Effects of calcium and cyclic nucleotides on rat calcitonin and parathyroid hormone secretion.

Recently we developed a system for studying concurrent secretion of calcitonin (CT)and parathyroid hormone(PTH)in vitro from single rat thyroparathyroid gland complexes. In the present study, mechanisms involved in secretion of CT and PTH were explored by altering the medium [Ca ] and by using the Ca antagonist, verapamil. We also re-examined the idea that cyclic nucleotides may help regulate secretion of these hormones and attempted to determine if effects of cyclic nucleotides might be altered by changes in medium [Ca]. Thyroparathyroid glands from 8-day-old rats were incubated in serum-free medium for 8h, and CT and PTH levels in the medium were measured by radioimmunoassays. We show for the first time that: (1) although low [Ca] is well known to promote PTH release, some extracellular Ca is needed for PTH secretion to occur at a maximal rate; (2) inhibition of Ca entry into cells with verapamil mimics the effects of low medium Ca on both CT and PTH release; and (3) cyclic nucleotides may exert their effects on secretion of CT and PTH at least in part via effects on Ca entry into cells.     

Separation of two bone cell populations from fetal rat calvaria and a study of their responses to parathyroid hormone and calcitonin

Bone cells released from perinatal rat calvaria by digestion with clostridial peptidase were separated into two distinct populations (designated types B and C) by equilibrium density centrifugation on a two-step gradient of Percoll. They were extensively characterized by light and electron microscopy and for behaviour in culture, acid and alkaline phosphatase activity, collagen synthesis, collagenase secretion and adenylate cyclase response to parathyroid hormone (PTH) and calcitonin. Type C cells were predominantly large with up to seven nuclei and an unusual cytoplasmic appearance in cytocentrifuge preparations. They did not proliferate in culture and we have established culture conditions which prevented their overgrowth by contaminating proliferative cells. In culture these cells had low alkaline and high acid phosphatase and high aryl sulphatase activity, and synthesized little collagen. In contrast type B cells were mostly smaller and many had irregular cytoplasmic projections. In culture they became polygonal in shape, proliferated rapidly, and reached confluence in 4-5 days. These were low in aryl sulphatase and acid phosphatase, high in alkaline phosphatase activity, and synthesized labelled collagen actively with [3H]proline and ascorbic acid included in the culture medium. The two cell population were found to differ in culture in two important further respects. First, the type C cells showed an adenylate cyclase response to calcitonin but not to PTH, while the converse was true for type B cells; this was so over at least a 20-fold range of isobutylmethyl xanthine concentration. Secondly, type C cells in culture secreted an active collagenolytic enzyme. Type B cells secreted much lower levels of a predominantly latent collagenase which required activation by mersalyl. Co-culture of type C and type B cells led to a marked reduction in the content of active collagenase in the culture medium.