Effect of calcium on cyclic nucleotide phosphodiesterase in parathyroid tissue

The hydrolysis of cyclic AMP and cyclic GMP by homogenates of normal bovine parathyroid gland and human parathyroid adenomas was decreased by EGTA. When supernatants were chromatographed on DEAE-cellulose it was found that sheep brain calmodulin in the presence of calcium stimulated cyclic AMP and cyclic GMP phosphodiesterase activity. The response to calmodulin in two human parathyroid adenomas was less than that in normal bovine parathyroid. Calmodulin was detected in heat-treated supernatants of 11 parathyroid adenomas by its ability to activate calmodulin-free sheep brain phosphodiesterase. The results suggest a role for calcium in the hydrolysis of cyclic nucleotides in parathyroid tissue.     

Influence of parathyroid status of rats on renal tubular handling of adenosine 3'5'-monophosphate: a micropuncture study

In order to correlate cyclic AMP handling by the nephron to the parathyroid status, clearance and micropuncture experiments were performed in rats with intact parathyroid glands, or immediately after parathyroidectomy, or six days after parathyroidectomy. In intact animals cyclic AMP urinary excretion was about twice the filtered load and the tubular addition of the nucleotide was achieved at the end of the accessible proximal tubule. In acutely parathyroidectomized rats cyclic AMP urinary excretion was not different from the filtered load and no proximal tubular addition was detected at the late accessible proximal tubule. In chronically parathyroidectomized animals urinary excretion of cyclic AMP was not different from the filtered load, nevertheless a proximal tubular addition of the nucleotide was observed, similar in magnitude to that of intact rats. The data afford a direct evidence that the convoluted proximal tubule is the major site of cyclic AMP tubular addition, confirm that this addition disappears immediately after parathyroidectomy, but indicate that it re-occurs in chronic parathyroidectomy.     

Regulation of secretion of parathormone and secretory protein-I from separate intracellular pools by calcium, dibutyryl cyclic AMP, and (1)- isoproterenol

Dispersed porcine parathyroid cells were incubated at calcium concentrations between 0.5 and 3.0 mM in the presence of 3H- or 14C- amino acids to label newly synthesized parathormone. Up to four times more hormone was secreted at the lower calcium concentration but its specific radioactivity, from 30 to 50 times that of the intracellular pool, did not change. Dibutyrl cyclic AMP doubled immunoactive parathormone secretion at each calcium concentration, but there was no increase in secretion of radioactive hormone if labeled amino acids and secretagogue were added simultaneously. Similarly, when the intracellular pool of parathormone was prelabeled with 3H-amino acids and then the cells were incubated in 14C-amino acids and dibutyryl cyclic AMP, the entire increase in hormone secreted was derived from the prelabeled pool. (1)-isoproterenol increased intracellular cyclic AMP and acted on hormone secretion in a manner indistinguishable from dibutyryl cyclic AMP. In similar double-label experiments dibutyryl cyclic AMP preferentially enhanced secretion of secretory protein-I, a calcium-regulated protein of the parathyroid of unknown function. Calcium, alone, inhibited the intracellular level of cyclic AMP in a concentration-dependent fashion. These data are consistent with the existence in the parathyroid cell preparation of two hormone and secretory protein pools that may be individually recruitable--one consisting of most recently synthesized protein, the other consisting of older "storage" protein. The data do not allow one to decide whether the two pools coexist within individual cells or whether, instead, they exist in separate cells of the dispersed gland preparation.     

Localization of cell groups sensitive to parathyroid hormone and calcitonin in rat skeletal tissue.

The level of cyclic AMP in various fractions of rat skeletal tissue was measured after in vitro or in vivo administration of parathyroid hormone and calcitonin. Incubations of bone fractions prepared from young (5 weeks of age thyroparathyroidectomized rats revealed that both parathyroid hormone and calcitonin increased the cyclic AMP level in fractions of epiphysis, metaphysis and marrow cells. Cyclic AMP accumulation in incubated perisoteum and diaphysis were induced solely by parathyroid hormone. In in vivo experiments the cyclic AMP level in the tibia of the thyroparathyroidectomized rat was increased by infusion of either parathyroid hormone or calcitonin, and the simultaneous administration of each maximally effective dose of the two hormones exhibited an additive effect. Within 2 min, parathyroid hormone infusion caused an elevation of cyclic AMP content in periosteum and metaphysis. Rapid increase of cyclic AMP in the metaphysis was also induced by calcitonin, and the effect of the two hormones on cyclic AMP accumulation in this fraction was additive. Small but significant increase of cyclic AMP in the diaphysis was detected at 5 min after the administration of parathyroid hormone. Calcitonin infusion did not show any consistent effects on periosteum and diaphysis.     

Hormone-specific responses and biosynthesis of sulfolipids in cell lines derived from mammalian kidney.

The established cell lines isolated from mammalian kidney were characterized by its receptor activities against hormones and the ability to synthesize sulfolipids localized in the renal tubule. The level of 3':5'-cyclic AMP in JTC-12.P3 (monkey kidney) cells increased in 2 min as much as 2.5-5-fold on activation with 1.0 unit/ml of bovine parathyroid hormone or 1.9 units/ml of synthetic parathyroid hormone (1-34) resulting in intracellular cyclic AMP concentration of more than 40 pmol/mg protein. Prostaglandin E1 (14 micronM) and isopropylnorepinephrine (10 micronM) were also found to increase the concentration of cyclic AMP by more than 30- and 9-fold, respectively. Addition in medium of calcitonin, arginine vasopressin, adrenocorticotropic hormone and glucagon caused no significant changes of cyclic AMP level in the cell. In contrast, MDCK, a cell line isolated from canine kidney, reacted to arginine vasopressin, isopropylnorepinephrine and prostaglandin E1 and only slightly to parathyroid hormone. MDBK cell line derived from bovine kidney or fibroblast cell lines from rat lung and guinea pig kidney did not react to any of the hormones specific to kidney, i.e. arginine vasopressin, calcitonin or parathyroid hormone in the presence of theophylline. However, in the presence of 2 mM isobutylmethylxanthine, small but significant elevation of cellular cyclic AMP levels in response to calcitonin, arginine vasopressin, isopropylnorepinephrine and prostaglandin E1 was observed. The cell lines JTC-12, MDCK and MDBK, when incubated with H235SO4, incorporated the isotope into sulfolipids assigned as sulfatides and ceramide dihexoside sulfate or in MDCK also into cholesterol sulfate. The results suggested that JTC-12, MDCK and MDBK cell lines are epithelial origin and also JTC-12 and MDCK originated most probably from renal tubular cells of cortex and medulla, respectively.     

Regulation of the receptor-mediated cyclic AMP response of kidney to parathyroid hormone in the vitamin D-deficient rat

Rats fed a diet deficient in vitamin D were found to exhibit a refractory cyclic AMP response of kidney slices to parathyroid hormone and a marked decrease in membrane parathyroid hormone-dependent adenylate cyclase activity. Both the characteristic calcium deficiency (hypocalcemia) and secondary elevation of circulating parathyroid hormone appeared before the first noticeable decrease in hormone-dependent enzyme activity. After repletion of D-deficient rats with vitamin D₂, we found that serum calcium and parathyroid hormone were both restored to normal levels before the depressed enzyme response to the hormone was reversed. Moreover, infusion of parathyroid hormone into vitamin D-replete rats led to a marked reduction in parathyroid hormone-dependent adenylate cyclase activity, which was partly restored to control level 3 hours after discontinuing the hormone infusion. Taken as a whole, this study suggests that the elevated endogenous parathyroid hormone in the vitamin D-deficient rat is involved in the “down-regulation” of renal cyclic AMP responsiveness to the hormone. However, these experiments do not rule out the possibility that calcium deficiency and/or vitamin D per se participate in the regulation of the renal cyclic AMP response to parathyroid hormone.     

Effects of parathyroid hormone on cyclic AMP, cyclic GMP, and efflux of calcium in isolated renal tubules.

The effects of parathyroid hormone (PTH) on concentrations of cyclic AMP and cyclic GMP were investigated in isolated renal cortical tubules from hamsters. Efflux of 45Ca from tubules was compared to temporal changes in both cyclic nucleotide concentrations. A rapid increase in cyclic AMP occurred following addition of PTH which was maximal by 1 min but decreased over the next 4 min period. Cyclic GMP concentrations were not significantly altered at 1 min but increased between 1 and 5 min from basal levels. Concentrations of both nucleotides remained significantly elevated from basal levels between 5 and 15 min following PTH. Efflux of 45Ca was increased by PTH with time-course changes closely paralleling changes in cyclic GMP concentrations. Changes in both cyclic AMP and cyclic GMP were related to PTH concentrations of the incubation media and were increased by addition of theophylline. Increasing the calcium concentration from 1 to 3 mM did not significantly alter the effect of PTH on cyclic AMP, however, cyclic GMP concentrations were further increased.     

Mode of action of somatostatin in inhibiting parathyroid hormone secretion

Effects of somatostatin on basal and low calcium-, isoproterenol- or dibutryl cyclic AMP (DBcAMP)-stimulated parathyroid hormone (PTH) secretion were evaluated in vitro with bovine parathyroid tissue. Low calcium, isoproterenol or DBcAMP alone significantly stimulated PTH secretion. Somatostatin 1 or 4 microgram/ml significantly inhibited these stimulated PTH secretions. Inhibition of isoproterenol-stimulated PTH secretion was more complete than was the inhibition of low calcium- or DBcAMP-stimulated secretion. The studies indicate that somatostatin inhibits PTH secretion by an action distal to cAMP generation. The more complete inhibition of isoproterenol-stimulated PTH secretion suggests that somatostatin may also have additional effects on or proximal to the formation of cyclic AMP.     

Hormone-specific responses and biosynthesis of sulfolipids in cell lines derived from mammalian kidney

The established cell lines isolated from mammalian kidney were characterized by its receptor activities against hormones and the ability to synthesize sulfolipids localized in the renal tubule.The level of 3′: 5′-cyclic AMP in JTC-12.P3 (monkey kidney) cells increased in 2 min as much as 2.5–5-fold on activation with 1.0 unit/ml of bovine parathyroid hormone or 1.9 units/ml of synthetic parathyroid hormone (1–34) resulting in intracellular cyclic AMP concentration of more than 40 pmol/mg protein. Prostaglandin E₁ (14 μM) and isopropylnorepinephrine (10 μM) were also found to increase the concentration of cyclic AMP by more than 30- and 9-fold, respectively. Addition in medium of calcitonin, arginine vasopressin, adrenocorticotropic hormone and glucagon caused no significant changes of cyclic AMP level in the cell.In contrast, MDCK, a cell line isolated from canine kidney, reacted to arginine vasopressin, isopropylnorepinephrine and prostaglandin E₁ and only slightly to parathyroid hormone. MDBK cell line derived from bovine kidney or fibroblast cell lines from rat lung and guinea pig kidney did not react to any of the hormones specific to kidney, i.e. arginine vasopressin, calcitonin or parathyroid hormone in the presence of theophylline. However, in the presence of 2 mM isobutylmethylxanthine, small but significant elevation of cellular cyclic AMP levels in response to calcitonin, arginine vasopressin, isopropylnorepinephrine and prostaglandin E₁ was observed.The cell lines JTC-12, MDCK and MDBK, when incubated with H₂³⁵SO₄, incorporated the isotope into sulfolipids assigned as sulfatides and ceramide dihexoside sulfate or in MDCK also into cholesterol sulfate.The results suggested that JTC-12, MDCK and MDBK cell lines are epithelial origin and also JTC-12 and MDCK originated most probably from renal tubular cells of cortex and medulla, respectively.     

Correlation between cyclic AMP levels and calcium efflux in isolated renal cortical tubules.

Isolated renal cortical tubules from male hamsters were utilized to examine the possible relationship between cyclic AMP (cAMP) and efflux of calcium. Both parathyroid hormone (PTH) and prostaglandin E1 (PGE1) produced dose-related increases in cAMP levels and calcium efflux from isolated tubules. Maximal concentrations of both hormones resulted in changes in cAMP which were 6 fold greater and changes in calcium efflux which were 2 fold greater with PGE1 than with PTH. Effects of sub-maximal amounts of either hormone on both cAMP and calcium efflux were potentiated to tubule incubations resulted in increases in tissue-associated cAMP over the same degree by inclusion of methyl-isobutylxanthine (MIX). Addition of either exogenous cAMP or dibutyryl cAMP (db-cAMP) produced dose-related increases in calcium efflux which occurred more rapidly with db-cAMP than with cAMP. Increasing amounts of cAMP added to the same concentration range resulting in increases in calcium efflux. Addition of 2', 3' cyclic AMP, 5'AMP or db-cyclic GMP had no significant effect on calcium efflux while 3', 5' cyclic CMP significantly reduced this response. The results indicate that cAMP increases efflux of calcium from renal tubules and may play a central role in hormone-dependent transport of this ion.     

Effect of imidazole on renal gluconeogenesis

The metabolic effects of imidazole were tested in rat renal cortex. Imidazole enhanced the activity of renal cortical phosphodiesterase in vitro. Imidazole inhibited glucose production in a dose-dependent fashion from a variety of substrates in the gluconeogenic pathway proximal to the triose phosphates. The stimulation in renal gluconeogenesis resulting from isoproterenol and parathyroid hormone was inhibited by imidazole. These changes correlated with an inhibition of the augmented levels of renal cortical cyclic AMP levels produced by these hormones. These studies indicate that imidazole is an effective activator of phosphodiesterase in intact renal cells and lend further support to the suggestion that the stimulation of renal gluconeogenesis produced by isoproterenol and parathyroid hormone is mediated by a release of cyclic AMP.     

Evidence for a Calcium/Calmodulin Involvement in Density-Dependent Melanogenesis in Murine B16 Melanoma Cells

Previous work from our laboratory has shown that both cyclic AMP and calcium/calmodulin appear to be involved in the regulation of melanogenesis in murine B16 melanoma cells. In these cells as in murine Cloudman S91 cells, melanogenic responsiveness to melanocyte-stimulating hormone (MSH) varies with cell density in culture. Our objective in this study was to learn more about the intracellular systems involved in the control of melanogenesis, particularly the role played by calcium. The melanogenic response to αMSH was compared to the response to drugs affecting intracellular free calcium and calmodulin over a range of cell densities in B16F1 cells. αMSH-stimulated melanin production was extremely density-dependent but αMSH-stimulated cyclic AMP production was independent of cell density. The melanogenic response to agents that increased intracellular calcium (A23187) or inhibited intracellular calmodulin varied with cell density. A drug (TMB8) that lowered intracellular free calcium, however, increased melanogenesis independently of cell density. At high cell density it was found that an elevation in calcium decreased melanogenesis, whereas agents that reduced calcium or inhibited calmodulin activity increased melanogenesis. At low cell density, however, the inhibitory response to A23187 was lost and in some experiments even stimulated melanogenesis. These data suggest that the calcium/calmodulin signalling system has an inhibitory influence on melanogenesis, and its expression, which depends upon cell density, may also modulate the response to stimulatory agents such as αMSH.     

Evidence for a calcium/calmodulin involvement in density-dependent melanogenesis in murine B16 melanoma cells.

Previous work from our laboratory has shown that both cyclic AMP and calcium/calmodulin appear to be involved in the regulation of melanogenesis in murine B16 melanoma cells. In these cells as in murine Cloudman S91 cells, melanogenic responsiveness to melanocyte-stimulating hormone (MSH) varies with cell density in culture. Our objective in this study was to learn more about the intracellular systems involved in the control of melanogenesis, particularly the role played by calcium. The melanogenic response to alpha MSH was compared to the response to drugs affecting intracellular free calcium and calmodulin over a range of cell densities in B16F1 cells. alpha MSH-stimulated melanin production was extremely density-dependent but alpha MSH-stimulated cyclic AMP production was independent of cell density. The melanogenic response to agents that increased intracellular calcium (A23187) or inhibited intracellular calmodulin varied with cell density. A drug (TMB8) that lowered intracellular free calcium, however, increased melanogenesis independently of cell density. At high cell density it was found that an elevation in calcium decreased melanogenesis, whereas agents that reduced calcium or inhibited calmodulin activity increased melanogenesis. At low cell density, however, the inhibitory response to A23187 was lost and in some experiments even stimulated melanogenesis. These data suggest that the calcium/calmodulin signalling system has an inhibitory influence on melanogenesis, and its expression, which depends upon cell density, may also modulate the response to stimulatory agents such as alpha MSH.     

Effect of imidazole on renal gluconeogenesis.

The metabolic effects of imidazole were tested in rat renal cortex. Imidazole enhanced the activity of renal cortical phosphodiesterase in vitro. Imidazole inhibited glucose production in a dose-dependent fashion from a variety of substrates in the gluconeogenic pathway proximal to the triose phsophates. The stimulation in renal gluconeogenesis resulting from isoproterenol and parathyroid hormone was inhibited by imidazole. These changes correlated with an inhibition of the augmented levels of renal cortical cyclic AMP levels produced by these hormones. These studies indicate that imidazole is an effective activator of phosphodiesterase in intact renal cells and lend further support to the suggestion that the stimulation of renal gluconeogenesis produced by isoproterenol and parathyroid hormone is mediated by a release of cyclic AMP.     

Calcium Dependence of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Accumulation in Rat Hippocampal Slices

Previous work has shown that incubation of hippocampal slices in medium without added calcium markedly attenuates the capacity of vasoactive intestinal peptide (VIP) to elevate cyclic AMP levels. The present studies examined the mechanism that confers calcium dependence on VIP stimulation of cyclic AMP accumulation in hippocampal slices. Calcium dependence was apparent immediately on slice preparation and was reversible only if calcium ions were added back very early during slice incubation (within 5 min). The cyclic AMP response to VIP was not abolished by preincubating slices in 100 microM adenosine, suggesting that calcium-dependent, VIP-induced release of adenosine does not mediate VIP elevation of cyclic AMP. VIP-stimulated cyclic AMP accumulation was not decreased by agents that block calcium influx (verapamil, nifedipine, magnesium ions), or by calmodulin antagonists (trifluoperazine, calmidozolium). In fact both verapamil (100 microM) and magnesium (14 mM) augmented VIP stimulation of cyclic AMP generation. Incubation of slices with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) did not affect VIP activation of cyclic AMP accumulation if slices were incubated without added calcium, but MIX did enhance VIP elevation of cyclic AMP content in slices incubated with calcium. Thus calcium dependence of the cyclic AMP response to VIP in hippocampal slices is unlikely to result from VIP-dependent calcium influx, from interactions with calmodulin, or from calcium-inhibited phosphodiesterase(s).(ABSTRACT TRUNCATED AT 250 WORDS)     

The roles of calcium and cyclic AMP in the stimulatory action of parathyroid hormone on thymic lymphocyte proliferation

Both the parathyroid hormone (PTH) and the calcium ion increase the cellular content of cyclic adenosine 3′,5′-monophosphate (cyclic AMP), promote the initiation of deoxyribonucleic acid synthesis and stimulate the proliferation of rat thymocytes maintained in vitro. The ability of cyclic AMP to serve as the mediator of the mitogenic actions of both PTH and calcium is established by the fact that cyclic AMP itself stimulates cell proliferation in the absence of PTH and extracellular calcium. Neither PTH nor calcium appear to raise the cellular cyclic AMP level by increasing the nucleotide's synthesis by adenylate cyclase (formerly adenyl cyclase); PTH concentrations as high as 50 μg per ml of medium do not increase the enzyme's activity (in the presence or absence of calcium) and mitogenic calcium concentrations inhibit it. PTH also does not directly affect isolated thymocyte phosphodiesterase, but mitogenic calcium levels inhibit the enzyme's activity. Additional experiments show that it is calcium which raises the cyclic AMP level in cells treated with PTH, and some possible calcium-mediated mechanisms by which the hormone could elevate the cellular cyclic AMP levels are discussed. Thus, the mitogenic action of PTH is primarily mediated by calcium while cyclic AMP is the ultimate implementor of the hormonal action. However, calcium has a dual role and evidence is presented which indicates that besides raising the cellular cyclic AMP level, it also controls the operation of cyclic AMP's mitogenic end-reaction.     

Stimulation of cellular autophagy by parathyroid hormone and cyclic adenosine 3′,5′: Monophosphate in isolated tubular fragments from the rat’s kidney cortex

Tubular fragments isolated from the cortex of the rat's kidney were qualitatively and quantitatively investigated with the electron microscope. The tubules frequently burst open and became "inverted" in such a way that the rarefied brush border now formed the outer circumference. By morphometry a decrease of the average cell volume in the proximal tubular fragments was ascertained. This was mostly caused by a loss of cytoplasmic ground substance, endoplasmic reticulum and ribosomes. Cytoplasmic herniations of the basal surface, filled with free ribosomes, suggested a partial shedding of the protein synthesizing apparatus. The number of autophagic vacuoles (AV) per unit area of cytoplasm was determined in proximal tubular fragments. After isolation alone, without further incubation, the number of AV was as low as the number found in an earlier study in proximal tubular cells in situ during the diurnal minimum. After control incubation the number of AV increased to about the mean value of the AV found in cells in situ during the whole diurnal cycle. By comparison with the control incubation the number of AV increased by a factor of 1.6 to 1.7, if cyclic adenosine 3',5': monophosphate (cyclic AMP) or parathyroid hormone (PTH) were added to the incubation-medium; it now reached about the number of AV found in situ during the diurnal maximum. The increase in the number of AV paralleled that of the production of ammonia and glucose from endogenous sources under the influence of cyclic AMP and PTH. This suggests that the breakdown of cytoplasmic components by cellular autophagy could be functionally related to gluconeogenesis. A quantitative comparison between the measured production of ammonia and glucose indicates, however, that in the system of isolated tubular fragments there may exist other mechanisms of degradation, and of the provision of substrates for gluconeogenesis, than cellular autophagy only.     

NMDA Receptor Activation Increases Cyclic AMP in Area CA1 of the Hippocampus via Calcium/Calmodulin Stimulation of Adenylyl Cyclase

Abstract: We observed previously that activation of N -methyl- d -aspartate (NMDA) receptors in area CA1 of the hippocampus, through either NMDA application or long-term potentiation (LTP)-inducing high-frequency stimulation (HFS), results in an increase in cyclic AMP. In the present study, we performed experiments to determine the mechanism by which NMDA receptor activation causes this increase in cyclic AMP. As the NMDA receptor-mediated increase in cyclic AMP is dependent upon extracellular calcium, we hypothesized that NMDA receptors are coupled to adenylyl cyclase (AC) via calcium/calmodulin. In membranes prepared from area CA1, AC was stimulated by calcium in the presence of calmodulin, and the effect of calcium/calmodulin on AC in membranes was blocked by the calmodulin antagonists N -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and trifluopera-zine (TFP). In intact hippocampal slices, W-7 and TFP blocked the increase in cyclic AMP levels caused by both NMDA application and HFS of Schaffer collateral fibers. Exposure of hippocampal slices to elevated extracellular potassium to induce calcium influx also caused increased cyclic AMP levels; the increase in cyclic AMP caused by high potassium was also blocked by W-7 and TFP. These data support the hypothesis that NMDA receptor activation is positively coupled to AC via calcium/calmodulin and are consistent with a role for cyclic AMP metabolism in the induction of NMDA receptor-dependent LTP in area CA1 of the hippocampus.     

Parathyroid hormone-induced changes in cyclic nucleotide levels during relaxation of the rabbit [correction of rat] aorta

Parathyroid hormone is a potent vasodilator in vivo and relaxes vascular tissue in vitro. Since parathyroid hormone action in kidney and bone is thought to be mediated by stimulation of cellular cyclic AMP production, the present study was designed to monitor changes in cyclic AMP and cyclic GMP in vascular tissue during relaxation by parathyroid hormone. Rabbit aortic strips were quick-frozen at various times after exposure to parathyroid hormone and the percent relaxation and cyclic nucleotide levels were determined. Cyclic AMP concentrations were elevated about 3-fold within 30 seconds after treatment with hormone. This corresponded to a 10% relaxation of the norepinephrine-contracted tissue. After five minutes, cyclic AMP was still elevated 2-fold above basal and the relaxation response was maximal (36%). The cyclic AMP and relaxation responses to parathyroid hormone were markedly potentiated by forskolin or methylisobutylxanthine. Parathyroid hormone produced a small but significant increase in cyclic GMP concentrations only at early time points whereas sodium nitroprusside substantially increased cyclic GMP and relaxed the strips at all times studied. The increase in cyclic AMP levels after exposure to parathyroid hormone occurred prior to or coincident with the onset of relaxation of the aortic strips. These findings are supportive of the hypothesis that the vascular actions of parathyroid hormone involve cyclic AMP.     

Microvillar elongation following parthenogenetic activation of sea urchin eggs

Parthenogenetic activation of unfertilized sea urchin eggs with ammonium chloride at pH 8.0 resulted in a slow, but dramatic, reorganization of surface microvilli in four species of sea urchin eggs. Following NH4Cl treatment, elongation of microvilli on the egg surface was observed concomitant with the formation of microfilament bundles within the microvillar cores. A minimum of 2 h of treatment was required for elongation and microfilament bundle formation to occur. The maintenance of elongated microvilli was pH-sensitive; removal of the activating agent resulted in the retraction of extended microvilli while readdition of NH4Cl caused microvilli to elongate again. Accompanying microvillar elongation in activated eggs, there was an increased calcium uptake as measured by 45Ca uptake. Blocking calcium uptake by incubation in lanthanum chloride or zero-calcium seawater containing 2 mM EGTA prevented microvillar elongation. These results suggested that elongation of microvilli following parthenogenetic activation by NH4Cl is pH- and calcium-dependent and is similar to that observed during normal fertilization.