Renal adenylate cyclase and the interrelationship between parathyroid hormone and vitamin D in the regulation of urinary phosphate and adenosine cyclic 3',5'-monophosphate excretion.

This study examined the role of cyclic AMP in the phosphaturic response to parathyroid hormone in vitamin D-deficient rats. Infusion of purified bovine parathyroid hormone (13.3 mug/h) into control, D-fed, or D-deficient, thyroparathyroidectomized rats produced a sixfold increase in renal phosphate and cyclic AMP excretion in D-fed rats, but only a two- to threefold increase in both parameters in D-deficient animals. Intravenous injection of parathyroid hormone over the dosage range from 1-50 mug/kg resulted in a dose-dependent increase in phosphate and cyclic AMP excretion with both D-fed and D-deficient thyroparathyroidectomized rats. However, the D-deficient rats responded to these injections of parathyroid hormone with a two- to threefold increase in both renal phosphate and cyclic AMP excretion at the highest dose of 50 mug/kg, whereas the D-fed animals' response was 35-fold and 11-fold over control excretion levels of phosphate and cyclic AMP, respectively. To directly examine the role of the renal cortical adenylate cyclase system in the blunted phosphaturic and urinary cyclic AMP responses to parathyroid hormone in D-deficient rats, we prepared a plasma membrane fraction enriched in this enzyme activity from the renal cortex of D-fed and D-deficient thyroparathyroidectomized rats. The renal cortical adenylate cyclase of D-deficient rats showed significantly (P less than 0.001) less activation by parathyroid hormone over the hormone concentration range from 0.3 to 7.0 mug/ml than was observed with the enzyme prepared from D-fed animals. Basal adenylate cyclase activity and the fluoride-stimulated enzyme activity were not altered by the state of D-deficiency. These experiments demonstrate that the blunted phosphaturic response to parathyroid hormone observed in D-deficient rats is associated with the reduced responsiveness of the renal cortical adenylate cyclase to the hormone. Moreover, the defect in the renal membrane adenylate cyclase system appears to be localized at the level of PTH binding to membrane receptors or, alternatively, at the level of transmission of the hormone-receptor binding signal to the catalytic moiety of this membrane enzyme.

A peptidomimetic antagonist of the alpha(v)beta3 integrin inhibits bone resorption in vitro and prevents osteoporosis in vivo.

Osteoclastic bone degradation requires intimacy between the matrix and the resorptive cell. While the precise role the integrin alpha(v)beta3 plays in the process is not yet understood, occupancy of the heterodimer by soluble ligand or by blocking antibody effectively inhibits bone resorption in vitro and in vivo, suggesting that alpha(v)beta3 blockade may prevent postmenopausal osteoporosis. Thus, we identified a synthetic chemical peptide mimetic, beta-[2-[[5-[(aminoiminomethyl)amino]-1-oxopentyl]amino]-1-+ ++oxoethyl]amino-3-pyridinepropanoic acid, bistrifluoroacetate (SC56631) based upon the alpha(v)beta3 ligand, Arg-Gly-Asp (RGD), which recognizes the isolated integrin, and its relative, alpha(v)beta5, as effectively as does the natural peptide. The mimetic dampens osteoclastic bone resorption in vitro and in vivo. Most importantly, intravenous administration of the mimetic prevents the 55% loss of trabecular bone sustained by rats within 6 wk of oophorectomy. Histological examination of bones taken from SC56631-treated, oophorectomized animals also demonstrates the compound's bone sparing properties and its capacity to decrease osteoclast number. Thus, an RGD mimetic prevents the rapid bone loss that accompanies estrogen withdrawal.

A peptidomimetic antagonist of the integrin alpha(v)beta3 inhibits Leydig cell tumor growth and the development of hypercalcemia of malignancy.

The integrin alpha(v)beta3 interacts with the arginine-glycine-aspartic acid (RGD) tripeptide recognition sequence of a variety of extracellular matrix proteins. Recent studies show that alpha(v)beta3 plays an important role in tumor-induced angiogenesis and tumor growth and that antagonists of alpha(v)beta3 inhibit angiogenic processes that include endothelial cell adhesion and migration. Consequently, we reasoned that an RGD-based peptidomimetic antagonist of alpha(v)beta3 might inhibit tumor angiogenesis and tumor growth in vivo. An RGD-peptidomimetic library was screened to identify antagonists of vitronectin binding to alpha(v)beta3, and the compounds chosen were modified to produce selective and potent inhibitors of alpha(v)beta3. One of these compounds, beta-[[2-2-[[[3-[(aminoiminomethyl)amino]-phenyl]carbonyl]amino]ac etyl]amino]-3,5-dichlorobenzenepropanoic acid (SC-68448), inhibited vitronectin binding to both alpha(v)beta3 and the closely related platelet receptor, alpha(IIb)beta3, in a dose-responsive manner. SC-68448 inhibited vitronectin binding to alpha(v)beta3 (IC50, 1 nM) and fibrinogen binding to the platelet receptor alpha(IIb)beta3 (IC50, >100 nM), demonstrating that SC-68448 was 100-fold more potent as an inhibitor of alpha(v)beta3 versus alpha(IIb)beta3. In cell-based studies, SC-68448 inhibited alpha(v)beta3-mediated endothelial cell proliferation in a dose-dependent manner but did not inhibit tumor cell proliferation, suggesting that effects on endothelial cell proliferation were not due to SC-68448-induced cytotoxicity. In accord with these results, SC-68448 inhibited angiogenesis in vivo in a basic fibroblast growth factor-induced rat corneal neovascularization model. A xenogeneic severe combined immune deficiency mouse/rat Leydig cell tumor model was developed for testing SC-68448 as an inhibitor of tumor growth in vivo. Rat Leydig cell tumors grew rapidly in severe combined immune deficiency mice and produced humoral hypercalcemia of malignancy. SC-68448 inhibited the growth of the tumors in mice by up to 80% and completely blocked the development of hypercalcemia. Together, these results demonstrate the feasibility of antitumor therapies based upon the development of nontoxic small molecule pharmacological antagonists of integrin alpha(v)beta3.

Identification and characterization of parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding.

Parathyroid hormone (PTH) receptors have been described in renal tissue from several species, but not in the rat. In this study, radioligand binding techniques were used to identify and characterize PTH receptors in rat kidney cortical membranes. The sulfur-free PTH analog [Nle8,18Tyr34]bovine PTH-(1-34)amide was iodinated using the iodogen method. This ligand was suitable for use in identifying PTH receptors in canine renal membranes, but not rat renal membranes. Synthetic, unsubstituted rat PTH-(1-34) was iodinated using the milder, lactoperoxidase technique and was purified by HPLC on a C8 column. [125I]rat PTH-(1-34) bound rapidly to both rat and dog renal membranes. At 22 degrees C reaction reached steady state within 20 minutes, and this level was maintained for at least 3 h. Specific binding was routinely greater than 90% for rat kidney and greater than 95% for dog kidney. Similar results were obtained at 4 degrees C with a longer time required to attain steady state (approximately 45 minutes). Binding was reversible as demonstrated by dissociation of bound ligand after either infinite dilution or displacement with excess nonradioactive PTH. Binding was saturable and of high affinity (rat kidney: Bmax = 2.3 pmol/mg protein, Kd = 3.1 nM, dog kidney: Bmax = 2.1 pmol/mg protein, Kd = 3.7 nM). Rat renal cortical adenylate cyclase activity was stimulated by rat PTH in a dose-dependent manner with an EC50 of 4 nM, a value in good agreement with the binding data. This study demonstrates the feasibility of identifying and characterizing parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding techniques.

Radioiodinated rat parathyroid hormone-(1-34) binds to its receptor on rat osteosarcoma cells in a manner consistent with two classes of binding sites.

Binding of 125I-labeled rat (r) PTH-(1-34) to ROS 17/2.8 osteoblastic bone cells and to membranes from these cells was examined. Competitive binding inhibition experiments were performed using unlabeled rPTH-(1-34) with particular emphasis on concentrations of peptide below 1 nM. In intact cells, binding of labeled rPTH-(1-34) was highly specific, and inhibition of binding by unlabeled ligand suggested the presence of two classes of binding sites, one with high affinity and low capacity (KD = 40 pM, approximately 20% of total binding sites) and the other with lower affinity and high capacity (KD = 2 nM, approximately 80% of the sites). Membranes prepared from ROS cells also exhibited a pattern of binding from competitive inhibition curves consistent with two distinct binding sites (KD = 30 pM and 6 nM). In intact ROS cells, cellular cAMP levels increased over the range of 10(-11)-10(-9) M rPTH-(1-34) with an ED50 intermediate between the two KD values (0.25 nM). These data suggest that osteoblastic bone cells possess two distinct classes of membrane receptors for PTH. Since the KD of the higher affinity site more closely approximates circulating concentrations of PTH, binding to this site may have physiologic relevance.

Binding of parathyroid hormone and parathyroid hormone-related protein to vascular smooth muscle of rabbit renal microvessels.

The humoral hypercalcemia of malignancy factor (also called PTH-related protein or PTHrp) has been shown to produce effects similar to PTH in the kidney, bone, and cardiovascular system. Binding of PTHrp and PTH has been characterized in renal and osseous tissues, but not in vascular tissue. We have attempted to characterize the interaction of both human PTHrp and rat PTH to renal microvessels as a model of vascular smooth muscle and in a renal tubule preparation from the same rabbit kidneys. Previous studies have shown the microvessel and tubule preparations to be distinct based upon morphological examination, differential enzyme markers, calcitonin and vasopressin-sensitive adenylate cyclase distribution, and different characteristics of guanine nucleotide and of oxidized PTH activation of the adenylate cyclases associated with the preparations. Human PTHrp and rat PTH were iodinated by standard techniques and purified by HPLC. Both ligands bound to microvessels and tubules in a saturable, specific manner, Maximal specific binding of either ligand was 65-75% in microvessels and 80-90% in renal tubules. The time courses of binding of both ligands were identical with steady state achieved within 20 min in the smooth muscle of microvessels and 15 min in the tubules at 22 C. In equilibrium competition binding experiments, bound 125I-PTHrp was displaced by both PTHrp and PTH in microvessels and tubules. Rat PTH displayed slightly higher affinity in microvessels and tubules than PTHrp. Identical results were obtained with 125I-PTH as ligand. Specificity of binding of PTHrp and PTH to both microvessels and tubules was excellent, with competition observed between the radioactive ligand and bovine and rat PTH, PTHrp, and the antagonists, [Nle8,18, Tyr34]bovine PTH and [Nle8,18, Tyr34]bovine PTH but not with several other peptides of unrelated structure. The only major difference in binding between microvessels and tubules was a smaller number of binding sites in microvessels compared to tubules. These results indicate that vascular tissue contains receptor sites for PTH and PTHrp as identified by radioligand binding techniques. These receptors are similar in characteristics to the receptors of renal tubular tissue. Both PTH and PTHrp appear to interact with the receptors of rabbit kidney microvessels and tubules.

Parathyroid hormone and calcium: interactions in the control of renin secretion in the isolated, nonfiltering rat kidney.

The present study was designed to characterize the interaction of calcium and PTH in the control of renin release in isolated rat kidneys perfused in a closed circuit at constant flow. Kidneys were rendered nonfiltering using low perfusion pressures (70 mm Hg) and a hyperoncotic perfusate (100 g/liter BSA). Under these conditions, differences in perfusion pressure were less than 9 mm Hg between control and PTH-treated kidneys over the 50 min of perfusion. In the absence of PTH, renin release was inversely correlated with ionized calcium (Ca2+) concentration, with the highest release of renin noted with 1 mM EGTA and no added calcium. Also, verapamil treatment markedly elevated renin release, even in the presence of 2 mM Ca2+. In contrast, renin secretion was strongly depressed by 20 nM BAY-K8644 in the perfusate. In medium containing normal calcium concentrations (1 mM Ca2+), rat PTH(1-34) induced a 2-fold greater renin accumulation than in the control, non-PTH-treated kidneys. Isoproterenol induced a 5-fold stimulation under the same conditions. In the 0 Ca2+/1 mM EGTA perfusion, PTH did not elevate renin secretion. Renin release in response to PTH in 2 mM Ca2+ was similar to that observed in the 1 mM Ca2+ perfusion. PTH also reversed the effects of BAY-K8644 to suppress renin release. In verapamil-treated kidneys, PTH failed to stimulate renin release. These results indicate that PTH stimulates renin release by a process independent of the baroreceptors and macula densa. The Ca2+ modulation of PTH-induced renin release is consistent with the reported ability of PTH to block calcium channels and relax vascular smooth muscle.

Endothelium-independent linkage of parathyroid hormone receptors of rat vascular tissue with increased adenosine 3',5'-monophosphate and relaxation of vascular smooth muscle.

We examined the mechanisms involved in the relaxation of rat vascular smooth muscle by PTH. PTH increased intracellular cAMP 10-fold in cultured vascular smooth muscle cells from rat aorta. Forskolin, methylisobutylxanthine, and papaverine all potentiated PTH action. The cAMP responses to PTH were not altered by concurrent addition of propranolol, phentolamine, atropine, or [Sar1,Ile8]angiotensin II. Only the synthetic PTH antagonist analog [Nle8,Nle18,Tyr34] bovine PTH-(3-34) inhibited the cAMP and vascular relaxation responses to PTH. Isoproterenol produced increases in intracellular cAMP and adenylate cyclase activity which were additive to those produced by PTH. In contracted rat aortic strips, PTH caused a dose-dependent relaxation which was not altered by removal of the vessel intima or treatment with nordihydroguaiaretic acid. Also, membrane preparations from intact aortas or aortas with the endothelium or adventitia removed displayed identical PTH-stimulated adenylate cyclase activities. These findings indicate that the relaxant action of PTH in rat aorta does not require an intact endothelium and results from a direct effect on the vessel medial layer. Relaxation appears to be mediated by a receptor unique for PTH which is linked to the adenylate cyclase of vascular smooth muscle cells.

Cardiovascular responsiveness to parathyroid hormone (PTH) and PTH-related protein in genetic hypertension.

Hypertension is often accompanied by abnormalities of calcium homeostasis, including hyperparathyroidism with reduced target organ responses to PTH in kidney and bone. Due to this association between PTH and hypertension and since PTH and the paracrine factor PTH-related protein (PTHrp) have both been shown to exert marked changes in cardiovascular activity, these actions of PTH and PTHrp were examined in spontaneously hypertensive rats (SHR) and in control normotensive Wistar-Kyoto rats (WKY). Fourteen-week-old SHR [systolic blood pressure (SBP), 201 +/- 4.4 mm Hg] and WKY (SBP, 141 +/- 2.5 mm Hg) were studied. Renal cortical membranes were prepared and assayed for radioligand binding with [125I]PTH-(1-34) and [125I]PTHrp-(1-34). There was no apparent alteration in the affinity of the binding sites to either peptide in the SHR, but specific binding in SHR renal tissue was only 60% of that observed in WKY tissue for both peptides. Serum immunoreactive PTH levels were 4-fold higher in SHR than WKY, while serum total calcium and 1,25-dihydroxyvitamin D3 levels were not different. The iv administration of both PTH and PTHrp produced dose-dependent reductions in SBP and increases in heart rate in conscious unrestrained SHR and WKY. Both peptides caused greater absolute reductions in blood pressure in SHR than in WKY. However, when the hypotensive response was normalized for the higher baseline pressure in the SHR, the blood pressure reductions caused by PTH and PTHrp were not different in SHR and WKY. Conversely, the chronotropic responses to PTH and PTHrp were lower in SHR compared to WKY. These findings indicate that the SHR exhibits elevated PTH levels, with a reduced number of renal PTH/PTHrp receptors and a depressed chronotropic response to either PTH or PTHrp. In contrast, the hypotensive response to PTH or PTHrp was not altered, indicating possible tissue-specific receptor subclasses or tissue-specific regulation of PTH and PTHrp receptors.

Hypotension and cardiac stimulation due to the parathyroid hormone-related protein, humoral hypercalcemia of malignancy factor.

Patients with humoral hypercalcemia of malignancy display markedly increased serum calcium levels, reduced blood pressure, and tachycardia. The causative agent, humoral hypercalcemia of malignancy factor [also called PTH-related protein (PTHrp)] has been shown to interact with PTH receptors in bone and kidney. We compared human PTHrp-(1-34) with rat PTH-(1-34) for the effects of each peptide on cardiovascular function in unrestrained conscious rats. Both PTHrp and PTH decreased blood pressure in a dose-dependent manner over the concentration range of 0.3-30 micrograms/kg. PTHrp was approximately 3-fold more potent than PTH, producing up to a 50 mm Hg decrease in pressure within 2 min at 10 micrograms/kg. Both peptides increased heart rate more than 70 beats/min at this dose. However, PTH appeared to exert greater efficacy and potency than PTHrp in increasing heart rate in vivo. In the isolated and perfused rat heart, PTHrp and PTH produced positive chronotropic and positive inotropic effects as well as increased coronary flow. PTHrp was more potent and more effective than PTH. The time courses of these effects in the perfused heart preparations indicated that both peptides produced maximal effects within 1 min, with all responses returning to baseline within 10 min. In isolated helical strips of rat aorta, PTHrp and PTH relaxed norepinephrine-contracted tissues in a concentration-dependent fashion. A functional endothelium was not required for the relaxing effects of either peptide. These studies indicate that PTHrp and PTH decrease blood pressure by relaxing vascular tissue in an endothelium-independent manner. Also, these peptides directly increased heart rate, contractility, and coronary flow. Since PTHrp has recently been found in normal human cells, these studies suggest the possibility of PTHrp as a regulator or modulator of cardiovascular function.

Synthetic alphaVbeta3 antagonists inhibit insulin-like growth factor-I-stimulated smooth muscle cell migration and replication.

Porcine aortic smooth cells respond to insulin-like growth factor-I (IGF-I) with increases in DNA synthesis and cell migration. Because ligand occupancy of the alphaVbeta3 integrin has been shown to be necessary for IGF-I to stimulate maximal increases in both processes, we determined whether synthetic alphaVbeta3 antagonists could inhibit IGF-I-stimulated actions on this cell type. Low-molecular-weight compounds that had been selected based on their ability to compete with vitronectin for binding to purified human alphaVbeta3 in vitro were analyzed for their ability to compete with 125I-kistrin (a known ligand for porcine alphaVbeta3) for binding to porcine alphaVbeta3. Nine compounds were screened, and five were found to be potent competitive inhibitors. The most potent compound, SC-69000, resulted in 88% competition at 10(-7) M and was nearly equipotent with echistatin. The compounds that were the most potent inhibitors of kistrin binding were tested for their capacity to inhibit the cell migration response to IGF-I. Three compounds caused between 81-88% inhibition of IGF-I-stimulated migration at 10(-7) M. To determine whether these compounds could inhibit other IGF-I-stimulated actions, their ability to inhibit IGF-I-stimulated [3H]-thymidine incorporation into DNA was analyzed. The four compounds that were the most potent inhibitors of cell migration also inhibited IGF-I-stimulated DNA replication. IGF-I stimulates the synthesis of IGF binding protein-5 by these cells. Preincubation with the four most active compounds also resulted in significant inhibition of the ability of IGF-I to stimulate IGF binding protein-5 synthesis. AlphaVbeta3 occupancy by the ligand vitronectin has been shown to enhance the capacity of IGF-I to activate its receptor tyrosine kinase. The four most active compounds were shown to inhibit IGF-I-stimulated IGF-I receptor autophosphorylation. These findings suggest that blockade of ligand occupancy of the alphaVbeta3 integrin globally inhibits several IGF-I-stimulated biologic actions and that synthetic inhibitors are very active in this regard. Because these compounds can be administered to whole animals, they should be very useful in determining whether blocking alphaVbeta3 occupancy in vivo results in alteration in responsiveness to IGF-I.

Antibody to beta3 integrin inhibits osteoclast-mediated bone resorption in the thyroparathyroidectomized rat.

The cell surface integrin, alphaVbeta3, is important for the attachment of osteoclasts to bone matrix and the subsequent resorption of bone. The present study was designed to determine the effects of F11, a monoclonal antibody to the rat beta3 subunit, on calcium mobilization in a rat model of bone resorption. Male Sprague Dawley rats became hypocalcemic within 18 h after thyroparathyroidectomy. Synthetic PTH-related protein (PTHrP(1-34)) administered to control rats caused serum calcium to return to normal. Anti-beta3 treatment of rats after thyroparathyroidectomy inhibited the calcemic response to PTHrP by 65%. Circulating F11 was biologically active as demonstrated by osteoclast retraction and by the inhibition of adenosine diphosphate-induced platelet aggregation via inhibition of the platelet integrin alphaIIbbeta3 in ex vivo assays. F11 antibody was localized by immunohistological staining to osteoclasts in long bones, suggesting that the mechanism of action of the antibody was via a direct effect upon osteoclasts. Echistatin and calcitonin also inhibited calcemic responses to PTHrP in this in vivo model, whereas an isotype-matched, control antibody was ineffective. These studies provide the first direct evidence in vivo that osteoclast-mediated bone resorption is regulated via beta3 integrin.

Insulin enhances pressor responses to norepinephrine in rat mesenteric vasculature.

Hyperinsulinemia and insulin resistance have been proposed to play a role in human and experimental hypertension. To characterize this relation further, we examined the pressor responses to periarterial nerve stimulation (PNS) and norepinephrine infusion in the isolated mesenteric vasculature of the normal rat before and after insulin (10, 100, and 1,000 microunits/ml) infusion. The pressor responses to PNS were similar before and after insulin, except at the highest dose of insulin (1,000 microunits/ml) and the highest frequency of PNS (10 Hz). However, insulin significantly increased the pressor responses to norepinephrine. This increase in responsiveness was evident at all doses of insulin studied. In contrast, insulin did not affect the pressor responses to either angiotension II or serotonin administration. The mechanism or mechanisms for the augmented pressor response to norepinephrine after insulin infusion remain to be determined. However, the selectivity of the response for norepinephrine and the absence of a marked pressor increase after PNS indicate that the mechanism probably involves either the alpha-receptor itself or an amplification of the postreceptor signal transduction. The role of chronic hyperinsulinemia and insulin resistance in the pathogenesis of essential hypertension requires further study.

Characterization of spontaneous metastasis in an aggressive breast carcinoma model using flow cytometry.

Studies of metastasis can be accelerated and provide more mechanistic information using cell lines which reproducibly and aggressively metastasize, and which are accurately and easily detected in tissues at all stages of the metastatic process. Although reporter proteins such as green fluorescent protein (GFP) and beta-galactosidase have improved the tracking of tumor cells in vivo, their measurement has often been limited to visual observation and manual counting. In this study, we exploited the highly sensitive and objective quantitation provided by flow cytometry to characterize, in detail, the sequence of events associated with orthotopic metastasis in a highly aggressive mouse model. Following stable transfection of the MDA-MB-435 breast carcinoma cell line with GFP, we utilized an in vivo selection process to isolate a variant exhibiting increased primary tumor growth and metastasis. As few as one fluorescent tumor cell per 200,000 host cells could be accurately detected in dissociated tissues by flow cytometry, allowing us to demonstrate that metastatic cells migrate to the lungs of SCID mice very early after orthotopic implantation. Tumor burden in lungs increased in a smooth continuous manner, until death approximately eight weeks later. Levels of circulating tumor cells in blood were also detectable at an early timepoint, but remained relatively low throughout the course of secondary tumor development in the lungs. Surgical removal of the primary tumor at various times after inoculation significantly affected lung tumor burden, supporting the concept that circulating tumor cells in blood inefficiently initiate distal metastases. Furthermore, the continuing contribution to metastasis by the primary tumor was independent of tumor mass. The combined characteristics of enhanced orthotopic metastasis and quantitative detection in blood and tissues will make this a useful new model for the characterization of the multi-stage progression of cancer, and the preclinical evaluation of anti-neoplastic therapies.

Regulation of skeletal muscle 6-phosphofructo-1-kinase during aging and development.

The purpose of this paper is to provide insight into the alterations of 6-phosphofructo-1-kinase (PFK) activity and isozyme types of rat skeletal muscle during development and aging. PFK isozymes are tetramers which may be comprised of one or any combination of the three subunit types, L, M, and C. The effects of fusion or terminal differentiation of cultured rat L6 myoblasts leading to formation of myotubles does not have a noticeable effect on total PFK activity. However, the amount of M-type subunit was increased; and the level of the C-type subunit decreased. These subunit changes caused shifts in the isozymic types. The ultimate effects of prenatal development of PFK were characterized in the near-term fetal muscle. This stage of development was accompanied by a significant loss of the C-type subunit and by two-fold increases in the L-type and M-type subunits which accounted for the 40% increase in total PFK activity. After birth, the M-type subunit increased dramatically as did the total PFK activity. Since the L-type and C-type subunits were gradually lost during the subsequent 3 weeks, the homotetramer of the M-type subunit (M4) was the only type which is present in mature muscle. M4 persisted as the only detectable form in skeletal muscle during the remainder of life, but the total PFK activity and amount of M4 decreased after 18 months of age. The decreased total PFK activity in aged skeletal muscle suggested that the expression of PFK genes may have reverted to an immature state when total PFK activity was lower. As shown by both the immunological analysis and direct quantification of subunit types, this clearly did not occur. That is, the loss of PFK activity in aged muscle is a consequence of decreased levels of the M-type subunit and not reappearance of other subunit types such as found in maturing muscle. Further, our examination of aged skeletal muscle indicates that no significant structural changes in M-type subunits had occurred and that inactive or partially active proteins which could crossreact with the M-type subunit were not detectable. It is suggested that the loss of M4 could cause a depression of the glycolytic rate leading to diminished ability of senile muscle to accommodate extreme energy demands.

Effect of dietary calcium supplementation on blood pressure and calciotropic hormones in mineralocorticoid-salt hypertension.

In order to determine the effect of dietary calcium supplementation on blood pressure and calciotropic hormones, we studied two groups (n = 12 each) of mineralocorticoid [deoxycorticosterone (DOC)]-salt hypertensive rats, one receiving a normal-calcium diet (0.6% calcium, as calcium carbonate) and the other a high-calcium diet (2.5% calcium), over an 8-week period. Dietary calcium supplementation significantly attenuated the rise in blood pressure. Serum ionized calcium concentrations were significantly decreased from baseline levels in both groups but tended to be higher among the calcium-supplemented rats. Serum concentrations of parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (1,25-D) were significantly higher in the DOC-salt rats than in normotensive rats fed normal rat chow [PTH: 49 +/- 4 versus 15 +/- 0.9 pg/ml (means +/- s.e.m.); 1,25-D: 108 +/- 7 versus 73 +/- 13 pg/ml, in DOC-salt and normotensive rats, respectively]. In the DOC-salt rats, dietary calcium supplementation did not significantly lower the elevated serum concentration of PTH (from 49 +/- 4 to 40 +/- 4 pg/ml; NS), but did significantly reduce that of 1,25-D (from 108 +/- 7 to 66 +/- 8 pg/ml; P less than 0.01). Since 1,25-D may increase vascular smooth muscle calcium uptake, dietary calcium supplementation may lower blood pressure in DOC-salt hypertension, in part, by suppressing 1,25-D.

Peptidomimetic antagonists of alphavbeta3 inhibit bone resorption by inhibiting osteoclast bone resorptive activity, not osteoclast adhesion to bone.

Osteoclasts are actively motile on bone surfaces and undergo alternating cycles of migration and resorption. Osteoclast interaction with the extracellular matrix plays a key role in the osteoclast resorptive process and a substantial body of evidence suggests that integrin receptors are important in osteoclast function. These integrin receptors bind to the Arg-Gly-Asp (RGD) sequence found in a variety of extracellular matrix proteins and it is well established that the interaction of osteoclast alpha v beta 3 integrin with the RGD motif within bone matrix proteins is important in osteoclast-mediated bone resorption. In this study, we characterized the effects of two synthetic peptidomimetic antagonists of alpha v beta 3, SC-56631 and SC-65811, on rabbit osteoclast adhesion to purified matrix proteins and bone, and on bone resorption in vitro. SC-56631 and SC-65811 are potent inhibitors of vitronectin binding to purified alpha v beta 3. Both SC-56631 and SC-65811 inhibited osteoclast adhesion to osteopontin- and vitronectin-coated surfaces and time-lapse video microscopy showed that osteoclasts rapidly retract from osteopontin-coated surfaces when exposed to SC-56631 and SC-65811. SC-56631 and SC-65811 blocked osteoclast-mediated bone resorption in a dose-responsive manner. Further analysis showed that SC-65811 and SC-56631 reduced the number of resorption pits produced per osteoclast and the average pit size. SC-65811 was a more potent inhibitor of bone resorption and the combination of reduced pit number and size led to a 90% inhibition of bone resorption. Surprisingly, however, osteoclasts treated with SC-65811, SC-56631 or the disintegrin echistatin, at concentrations that inhibit bone resorption did not inhibit osteoclast adhesion to bone. These results suggest that alphavbeta3 antagonists inhibited bone resorption by decreasing osteoclast bone resorptive activity or efficiency but not by inhibiting osteoclast adhesion to bone per se.

Increased cyclic AMP in cultured vascular smooth muscle cells and relaxation of aortic strips by parathyroid hormone.

The effects of parathyroid hormone (PTH) were examined in three model systems to determine the mechanism of PTH action in vascular tissue. Bovine parathyroid extract and synthetic bPTH-(1-34) relaxed norepinephrine-contracted rabbit aortic strips in a dose-dependent fashion. The ED50 was 33.1 nM (21.8-50.1 nM). The hypotensive diterpene, forskolin and the phosphodiesterase inhibitors, methylisobutylxanthine and papaverine, all greatly potentiated the relaxant action of PTH. Primary cultures of vascular smooth muscle cells isolated from rabbit and rat aorta and bovine pulmonary artery all responded to bPTH-(1-34) with 5- to 10-fold increases in intracellular cyclic AMP concentrations within 1 min. Again, this action of PTH was also markedly augmented (3-fold or greater) by methylisobutylxanthine, papaverine or forskolin. In addition, 1 microM bPTH-(1-34) stimulated adenylate cyclase activity in membrane preparations from vascular smooth muscle cells in the presence or absence of 100 microM GMPPNHP. These results indicate that PTH exerts direct relaxant actions on vascular smooth muscle and that cyclic AMP may be involved in the mechanism of PTH action in vascular tissue.

Manganese potentiation of nitric oxide-mediated vascular relaxation.

The half-life of nitric oxide (NO) and the relaxation of aortic-rings are enhanced by superoxide dismutase. Manganese and manganese-containing preparations have been reported to mimic superoxide dismutase activity. In the current study, manganese was tested for its ability to potentiate the activity of NO both in vitro and in vivo. Manganese relaxation of aortic segments was endothelium dependent as well as concentration dependent. Cyclic GMP concentrations in the segments were increased 2- and 4-fold with 5 and 300 microM manganese, respectively. N-Monomethyl-L-arginine pretreatment of aortic rings abolished the relaxation and cyclic GMP accumulation mediated by manganese. Infusion of manganese into conscious, restrained rats resulted in a decrease of blood pressure which was abolished by N-nitro-L-arginine pretreatment. Therefore, manganese may prolong the half-life of NO by a mechanism that mimics the action of superoxide dismutase resulting in potentiation of NO actions in vascular tissue.

Analogue separates biological effects of salmon calcitonin on brain and renal cortical membranes.

The conformation-activity relationship of salmon calcitonin in kidney and brain was investigated with regard to effects on membrane binding and adenylate cyclase activity. Since an amphipathic alpha-helical conformation on the calcitonin molecule is associated with high potency in lowering serum calcium, the activity of the parent peptide was compared to that of [Gly8, D-Arg24]des-Leu16-salmon calcitonin, a calcitonin analogue (CTA) with less helix forming potential. The results indicate that while salmon calcitonin possesses similar potency in brain and kidney, CTA is effective only in brain. Furthermore, CTA did not inhibit the binding of 125I-labeled human calcitonin gene-related peptide (HCGRP) to brain membranes. Our findings suggest that the specific binding and effects of salmon calcitonin on adenylate cyclase activity in brain do not depend on conformational features in the middle region of the molecule, although the alpha-helical structure in this region does appear to be an important property for salmon calcitonin binding to renal cortical membranes.