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Biomedical subjects

M R Hammerman

Publications and source records attributed to M R Hammerman.

At least 91 records · Page 5Linked to original sources
PubMed1989

Growth hormone directly stimulates gluconeogenesis in canine renal proximal tubule.

To characterize the action of growth hormone (GH) on gluconeogenesis in renal proximal tubule, glucose production was measured in suspensions of canine renal proximal tubular segments incubated with 1 mM L-alanine, 10 mM lactate, 1 mM succinate, and various concentrations (10(-11) to 10(-6) M) of recombinant bovine GH (bGH). Production of glucose increased as a function of time for 120 min. Bovine growth hormone (10(-6) M) increased glucose production at 120 min by 55 +/- 16%. Significant enhancement of glucose production occurred in suspensions of segments incubated with as little as 10(-10) M bGH. Half-maximal stimulation occurred at between 10(-9) and 10(-8) M. To ascertain whether these actions of bGH are mediated directly, we determined the effects of insulin-like growth factors (IGFs) I and II on glucose production. Addition of IGF-I to segments enhanced glucose production in a concentration-dependent manner. However, incubation with bGH did not induce measurable IGF-I production in the segments. In contrast to the action of IGF-I, IGF-II did not affect glucose production. We conclude that bGH acts directly on cells within proximal tubular segments to enhance gluconeogenesis. Stimulation of gluconeogenesis in vitro could reflect a counterregulatory action of GH exerted on renal proximal tubule in vivo.

Animals↗
PubMed1989

Dual regulation of insulin-like growth factor I expression during renal hypertrophy.

We examined the regulation of insulin-like growth factor I (IGF-I) in kidney during the renal hypertrophy produced by two different experimental models: growth hormone treatment of hypophysectomized rats and compensatory hypertrophy subsequent to unilateral nephrectomy. Immunostaining for IGF-I in collecting ducts was enhanced in kidneys from growth hormone-repleted hypophysectomized rats, and the levels of IGF-I mRNAs were increased. In compensatory hypertrophy, no enhancement of the intensity of immunostaining was observed in kidneys of nephrectomized rats until 5 days postnephrectomy, at which time immunostainable IGF-I was increased markedly in medullary collecting ducts of hypertrophied kidneys compared with kidneys from sham-operated animals. No difference in steady-state levels of any IGF-I mRNA species was detected in whole kidneys or in collecting ducts from nephrectomized or sham-operated rats at any time postnephrectomy. Our findings demonstrate an increase in both IGF-I mRNA and in immunostainable IGF-I in collecting duct in the setting of growth hormone-induced renal hypertrophy but suggest that other, possibly translational, mechanisms underlie the induction of IGF-I synthesis during compensatory hypertrophy.

Animals↗
PubMed1989

The growth hormone-insulin-like growth factor axis in kidney.

Growth hormone (GH) exerts a variety of metabolic and anabolic effects on skeletal and soft tissues including kidney. Some of these actions are mediated directly, whereas others result from GH-dependent synthesis and release of polypeptide growth factors designated insulin-like growth factors (IGFs). Receptors for GH are present in proximal tubule and GH directly stimulates gluconeogenesis at this site. IGF receptors are found in glomerulus and proximal tubule. Mechanisms for signal transduction by GH and IGFs have been characterized using proximal tubular basolateral membranes. IGFs regulate metabolic and transport processes in cultured glomerular mesangial cells and in isolated proximal tubular cells. IGF I is synthesized in cultured mesangial cells and is produced in a GH-dependent manner in cortical and medullary collecting duct. Evidence has accumulated that IGF I of renal origin functions as a paracrine growth factor in the settings of GH-induced hypertrophy and compensatory hypertrophy of the kidney, and in the setting of proximal tubular regeneration following ischemic injury. IGFs are embryonal mitogens and IGF II may act as a transforming agent for Wilms' tumor. Further characterization of the GH-IGF axis in kidney will provide additional insights into the roles of these peptides as regulators of renal function, growth, and development.

Amino Acid Sequence↗
PubMed1988

Insulin-like growth factor II stimulates production of inositol trisphosphate in proximal tubular basolateral membranes from canine kidney.

To determine whether insulin-like growth factor II (IGF-II) activates phospholipase C in the basolateral membrane of the renal proximal tubular cell, we incubated basolateral membranes isolated from canine kidney with rat IGF-II (rIGF-II) and measured levels of inositol trisphosphate (Ins-P3) in suspensions and of diacylglycerol extractable from the membranes. Incubation with rIGF-II increased levels of Ins-P3 and diacylglycerol in a concentration-dependent manner. Significant enhancement of Ins-P3 levels and extractable diacylglycerol occurred in suspensions incubated with as little as 10(-10) M rIGF-II. Elevated levels of Ins-P3 were measured after as little as 5 sec of incubation. Increases were no longer detectable after 45 sec of incubation, due to dephosphorylation of Ins-P3 in membrane suspensions. Incubation with either insulin or insulin-like growth factor I did not affect the level of Ins-P3. IGF-II-stimulated increases in Ins-P3 did not occur when basolateral membranes were suspended in the absence of free calcium. Increases were demonstrable in basolateral membrane suspensions in 0.1, 0.2, or 0.3 microM calcium, but not in 1.0 microM calcium. Inclusion of guanosine 5'-[gamma-thio]triphosphate in incubation mixtures did not increase levels of Ins-P3, nor did it enhance the action of rIGF-II in this regard. However, inclusion of guanosine 5'-[beta-thio]diphosphate inhibited rIGF-II stimulation of Ins-P3 production. In contrast to findings with basolateral membrane suspensions, incubation with rIGF-II did not increase levels of Ins-P3 in suspensions of isolated brush-border membranes. Our data are consistent with IGF-II-mediated activation of phospholipase C in isolated proximal tubular basolateral membranes. Such an action could reflect the mechanism by which the IGF-II "signal" is transmitted across the basolateral membrane of the renal proximal tubular cell and by which the actions of this peptide are mediated in renal and non-renal cells.

Cell Membrane↗
PubMed1988

Focal expression of insulin-like growth factor I in rat kidney collecting duct.

To address the question of insulin-like growth factor (IGF) I localization and synthesis in kidney, we used two complementary experimental approaches: immunohistochemistry of fixed paraffin-embedded rat kidney sections; and measurement of IGF I mRNA in isolated components of the rat nephron, using a highly sensitive and specific solution hybridization assay. Immunostainable IGF I was localized exclusively to principal cells of cortical and medullary collecting ducts. Administration of growth hormone to hypophysectomized rats for 8 d resulted in enhanced immunohistochemical staining of IGF I within collecting ducts, but no detectable IGF I in other portions of the nephron. The abundance of IGF I mRNA was 7-12-fold higher in isolated papillary collecting ducts than in proximal tubules or glomeruli, and was enriched 10-fold compared with whole kidney. Our data demonstrate colocalization of IGF I and IGF I mRNA in the collecting duct, consistent with focal expression of the IGF I gene at this site.

Animals↗
PubMed1988

Na+-independent D-glucose transport in rabbit renal basolateral membranes.

To define the mechanism by which glucose is transported across the basolateral membrane of the renal proximal tubular cell, we measured D-[14C]glucose uptake in basolateral membrane vesicles from rabbit kidney. Na+-dependent D-glucose transport, demonstrable in brush-border vesicles, could not be demonstrated in basolateral membrane vesicles. In the absence of Na+, the uptake of D-[14C]glucose in basolateral vesicles was more rapid than that of L-[3H]glucose over a concentration range of 1-50 mM. Subtraction of the latter from the former uptakes revealed a saturable process with apparent Km of 9.9 mM and Vmax of 0.80 nmol.mg protein-1.s-1. To characterize the transport component of D-glucose uptake in basolateral vesicles, we measured trans stimulation of 2 mM D-[14C]glucose entry in the absence of Na+. Trans stimulation could be effected by preloading basolateral vesicles with D-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose, but not with L-glucose or alpha-methyl-D-glucoside. Trans-stimulated D-[14C]glucose uptake was inhibited by 0.1 mM phloretin or cytochalasin B but not phlorizin. In contrast, Na+-dependent D-[14C]glucose transport in brush-border vesicles was inhibited by phlorizin but not phloretin or cytochalasin B. Our findings are consistent with the presence of a Na+-independent D-glucose transporter in the proximal tubular basolateral membrane with characteristics similar to those of transporters present in nonepithelial cells.

3-O-Methylglucose↗
PubMed1988

Parathyroid hormone stimulates ammoniagenesis in canine renal proximal tubular segments.

To determine whether parathyroid hormone (PTH) affects ammoniagenesis in renal proximal tubule, we measured ammonia production in suspensions of canine renal proximal tubular segments incubated with 10 mM L-glutamine in the absence or presence of 10(-7) M PTH. Productions of ammonia were linear functions of time for 120 min and averaged 231 +/- 55 and 311 +/- 67 mumol ammonia/g protein in the absence and presence of PTH, respectively. When measured over the range of 10(-11)-10(-7) M PTH, half-maximal stimulation of ammonia production occurred between 10(-10) and 10(-9) M PTH. Maximal production of ammonia was observed at 10(-8) M PTH. Incubation of proximal tubular segments with PTH increased levels of adenosine 3',5'-cyclic monophosphate (cAMP) in vitro. Ammonia production was significantly enhanced by incubation of segments with the cAMP analogue, 8-bromoadenosine 3',5'-cyclic monophosphate. PTH also increased ammonia production in segments incubated with 1 mM L-glutamine. We conclude that PTH stimulates ammonia production in canine renal proximal tubular segments. This effect appears to be mediated, at least in part, through cAMP in vitro. Such stimulation could reflect a direct action of PTH on the proximal tubule to enhance ammoniagenesis in vivo.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Calcium-activated phospholipase C associated with canine renal basolateral membranes.

To determine whether calcium-activated phospholipase C effects breakdown of phospholipids present in the basolateral membrane of the renal proximal tubular cell we incubated proximal tubular basolateral membranes isolated from dog kidney in the absence and presence of deoxycholate, and varied free calcium from 0 to 300 microM. Following incubations, lipids were extracted from membranes and separated using thin-layer chromatography. In the absence of deoxycholate, neither phosphatidylinositol nor diglyceride extractable from basolateral membranes changed significantly as calcium was increased. In the presence of deoxycholate, extractable phosphatidylinositol and diglyceride did not change significantly as free calcium was elevated from 0 to 0.03 microM, but phosphatidylinositol decreased and diglyceride increased progressively as 0.03-300 microM free calcium was included in incubations. Most of the increased extractable diglyceride could be accounted for by hydrolysis of phosphatidylinositol as reflected by decreased extractable phosphatidylinositol. Our findings suggest that calcium-activated phospholipase C effects breakdown of phospholipids present in the basolateral portion of the plasma membrane of the renal proximal tubular cell. It is possible that this enzyme plays a role in stimulating protein kinase c at this site.

Animals↗
PubMed1987

Phorbol esters inhibit ammoniagenesis and gluconeogenesis in proximal tubular segments.

To characterize the regulation of ammoniagenesis and gluconeogenesis in renal proximal tubule, ammonia and glucose productions were measured in suspensions of canine proximal tubular segments incubated with 10 mM L-glutamine. Productions were linear functions of time for 120 min and were decreased as extracellular pH was increased from 7.0 to 7.5 To ascertain whether activation of protein kinase c affects either process, we incubated segments with tumor-promoting phorbol esters, 12-O-tetradecanoylphorbol-13-acetate (TPA), or phorbol 12,13-dibutyrate, or with the inactive phorbol ester 4 alpha-phorbol. Ammoniagenesis and gluconeogenesis were inhibited by incubation with 10(-6) M of the two former compounds but not the latter compound. Inhibition of ammoniagenesis and gluconeogenesis occurred after incubation with as little as 10(-9) M phorbol 12,13-dibutyrate. Phorbol ester-induced inhibition was observed under conditions such that extracellular [Na+] was greater than intracellular [Na+], but not when extracellular [Na+] equaled intracellular [Na+], and was not observed in the presence of amiloride. Our findings are consistent with a role for protein kinase c in the control of ammoniagenesis and gluconeogenesis in proximal tubule. Such control could be mediated via stimulation of Na+-H+ exchange.

Ammonia↗
PubMed1987

Distribution of IGF receptors in the plasma membrane of proximal tubular cells.

To characterize the distribution of receptors for insulin-like growth factors I and II (IGF I and II) in the plasma membrane of the renal proximal tubular cell, we measured binding of 125I-labeled IGF I and 125I-labeled IGF II to proximal tubular basolateral and brush-border membranes and characterized IGF I-stimulated phosphorylation of detergent-solubilized membranes. 125I-IGF I bound primarily to a 135,000 relative molecular weight (Mr) protein and IGF II to a 260,000 Mr protein in isolated membranes. Binding of 125I-IGF I was severalfold greater in basolateral than in brush-border membranes. IGF I-stimulated phosphorylation of the 92,000 Mr beta-subunit of its receptor could be demonstrated only in basolateral membranes. These findings are consistent with an asymmetrical distribution of receptors for IGF I in the plasma membrane of the renal proximal tubular cell, localization being primarily on the basolateral side. In contrast, binding of 125I-IGF II to isolated basolateral and brush-border membranes was equivalent, suggesting that receptors for this peptide are distributed more symmetrically in the plasma membrane. Our findings suggest that the actions of IGF I in proximal tubule are mediated via interaction of circulating peptide with specific receptors in the basolateral membrane. However, our findings establish the potential for actions of IGF II to be exerted in proximal tubule via interaction with both basolateral and/or brush-border membrane receptors.

Animals↗
PubMed1987

Insulin stimulates ammoniagenesis in canine renal proximal tubular segments.

To characterize the effect of insulin on ammoniagenesis in renal proximal tubule, ammonia productions were measured in suspensions of canine renal proximal tubular segments incubated with 10 mM L-glutamine and varying concentrations of insulin. Productions of ammonia were linear functions of time for 120 min. Insulin (10(-6) M) increased ammonia production at 2 h by 34 +/- 5%. At the same time, gluconeogenesis, as measured by glucose production, was decreased by 16 +/- 2%. Significant enhancement of ammoniagenesis occurred in suspensions of segments incubated with as little as 10(-9) M insulin. Half-maximal stimulation occurred at between 10(-9) and 10(-8) M insulin. Oxidation of L-glutamine in cells within segments was also increased by insulin in a concentration-dependent manner. Insulin increased ammonia productions in segments incubated at pH 7.5 but not at 7.0. Under the former conditions, insulin enhanced ammoniagenesis in proximal tubular segments under conditions such that extracellular [Na+] was greater than intracellular [Na+], but not when extracellular [Na+] equaled intracellular [Na+]. We conclude that insulin stimulates ammonia production in suspensions of canine renal proximal tubular segments. Stimulation of ammonia production in vitro could reflect an action of insulin to enhance proximal tubular ammoniagenesis in vivo.

Ammonia↗
PubMed1987

Insulin-like growth factors and aging.

Since its proposal three decades ago, the evidence in favor of the somatomedin hypothesis has been compelling. It is clear that somatotrophic actions of growth hormone are mediated through generation of insulin-like peptides and interaction of these peptides with plasma membrane receptors on sensitive cells. It is possible that such actions result from effects of circulating insulin-like peptides and/or insulin-like peptides generated in proximity to their sites of action (autocrine or paracrine effects). Most or all of circulating somatomedin activity in humans can be accounted for by insulin-like growth factors I and II (IFGs I and II). These peptides have considerable structural homology with insulin but, unlike insulin, they circulate in tight, noncovalent association with specific carrier protein. Levels of circulating IGF I and IGF II are affected by growth hormone, but the former peptide is the more sensitive to growth hormone. Levels of circulating IGF I in humans are low at birth, rise progressively during childhood, and peak during midadolescence. The increase in stature that occurs normally during adolescence probably results from this increase in circulating IGF I. Following adolescence, levels of circulating IGF I fall progressively as a function of age. There is good evidence that the reduction in levels of circulating IGF I is related to decreased secretion of growth hormone that accompanies aging. Although it has been suggested that decreased function of the growth hormone-somatomedin axis may cause changes in anabolic indices that accompany the aging process, definitive proof for this hypothesis is lacking. In contrast to IGF I, circulating IGF II reaches "adult" levels early in childhood, and changes are relatively small as a function of increasing age. Counterparts of IGF I and IGF II are present in rats. Dynamics of the growth hormone-somatomedin axis are similar in rats and humans for IGF I. In contrast, levels of IGF II in rat fall precipitously following birth, suggesting a role for rat IGF II in fetal growth and development. The rat has been used as an experimental animal to define the role of the growth hormone-somatomedin axis in aging. As in human studies, no firm relationship between somatomedins and aging has been established in the rat.

Adult↗
PubMed1986

Multiplication-stimulating activity-induced alkalinization of canine renal proximal tubular cells.

The actions of a variety of polypeptide growth factors on isolated cells are thought to be initiated by stimulation of Na+-H+ exchange across the plasma membranes of the cells resulting in intracellular alkalinization. To determine whether insulin-like growth factors (IGFs) exert actions through such a mechanism, we incubated suspensions of canine renal proximal tubular segments with insulin or IGF I or with multiplication-stimulating activity (MSA)/IGF II. Changes in intracellular pH were detected by measurements of the distribution of [14C]5,5-dimethoxazolidine-2,4-dione. Incubation of segments with 10(-9) M MSA under conditions such that extracellular [Na+] greater than intracellular [Na+] effected intracellular alkalinization detectable within 1-2 min. Alkalinization was not observed under conditions where this gradient was not present. Alkalinization was not prevented by inclusion of 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid or 1 mM ouabain in incubations, but was inhibited by amiloride. Incubation of proximal tubular segments with as little as 10(-11) M MSA effected intracellular alkalinization. Incubation with as much as 10(-6) M insulin or IGF I did not. Our findings are consistent with an action of MSA/IGF II to stimulate Na+-H+ exchange across the plasma membrane of the renal proximal tubular cell. It is possible that the stimulation represents a mechanism by which actions of IGF II are initiated in growth factor-sensitive cells.

Animals↗
PubMed1986

Binding of IGF I and IGF I-stimulated phosphorylation in canine renal basolateral membranes.

To characterize the interaction of the renal proximal tubular cell with insulin like growth factor I (IGF I), we measured binding of 125I-IGF I to proximal tubular basolateral membranes from dog kidney and induced IGF I-stimulated phosphorylation of basolateral membranes. Specific binding of 125I-IGF I to basolateral membranes was observed that was half-maximal at between 10(-9) and 10(-8) M IGF I. 125I-IGF I was affinity cross-linked to a 135,000 Mr protein in basolateral membranes that was distinct from the alpha-subunit of the insulin receptor and from the IGF II receptor. IGF I-stimulated phosphorylation of a 92,000 Mr protein was effected in detergent-solubilized membranes incubated with 100 microM [gamma-32P]ATP. The 32P-labeled protein was distinct from the beta-subunit of the insulin receptor, the 32P phosphorylation of which was stimulated by insulin. We conclude that specific receptors for IGF I are present in the basolateral membrane of the renal proximal tubular cell. Physiological actions of IGF I at this nephron site may occur through the binding of this peptide circulating in plasma, to specific basolateral membrane receptors, followed by IGF I stimulated phosphorylation.

Animals↗
PubMed1986

Electrogenic Na+-independent Pi transport in canine renal basolateral membrane vesicles.

To define the mechanism by which Pi exists from the renal proximal tubular cell across the basolateral membrane, we measured 32Pi uptake in basolateral membrane vesicles from dog kidney in the absence of Na+. Preloading of basolateral vesicles with 2 mM Pi transstimulated 32Pi uptake, which is consistent with counterflow. We used measurements of transstimulation to quantitate the transport component of 32Pi uptake. Transstimulation of 32Pi uptake was inhibited less than 30% by concentrations of probenecid as high as 50 mM. In contrast, transstimulation of 35SO4(2-) uptake by intravesicular SO4(2-) was inhibited 92% by 5 mM probenecid. Preloading basolateral vesicles with SO4(2-) did not result in transstimulation of 32Pi uptake. Accumulation of 32Pi in basolateral vesicles above steady state was driven by a membrane potential (intravesicular positive), consistent with Na+-independent Pi transport being accompanied by the net transfer of negative charge across the membrane. We conclude that carrier-mediated, electrogenic Na+-independent 32Pi transport can be demonstrated in basolateral vesicles from dog kidney. This process appears to be mediated, at least in part, via a mechanism different from that by which SO4(2-) is transported. Electrogenic Na+-independent Pi transport may reflect one means by which Pi reabsorbed across the luminal membrane exists from the proximal tubular cell down an electrochemical gradient.

Animals↗
PubMed1986

Phorbol ester-induced alkalinization of canine renal proximal tubular cells.

To determine whether exposure of renal proximal tubular cells to tumor-promoting phorbol esters results in intracellular alkalinization, we incubated suspensions of proximal tubular segments isolated from dog kidney with these compounds. Changes in intracellular pH were detected by measurements of the distribution of [14C]5,5-dimethoxazolidine-2-4-dione (DMO). Incubations of segments with 10(-6) M of the tumor-promoting phorbol esters phorbol-12,13-dibutyrate or 12-O-tetradecanoylphorbol-13-acetate (TPA), under conditions such that extracellular [Na+] greater than intracellular [Na+], effected intracellular alkalinization detectable within 1-2 min. Incubation with the inactive phorbol ester 4 alpha-phorbol did not. Incubation with as little as 10(-10) M phorbol-12,13-dibutyrate effected intracellular alkalinization. Incubation with 10(-12) M phorbol-12,13-dibutyrate did not. Alkalinization was not observed under conditions such that extracellular [Na+] = intracellular [Na+]. Alkalinization could be prevented by inclusion of 10(-3) M amiloride in incubations. Our findings are consistent with an action of tumor-promoting phorbol esters to stimulate Na+-H+ exchange across the plasma membrane of the renal proximal tubular cell. It is possible that the stimulation reflects a mechanism by which hormones and/or growth factors exert actions in proximal tubule.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1986

Phosphorylation of type II cAMP-dependent protein kinase in renal brush border membranes.

We have previously demonstrated cAMP-dependent 32P phosphorylation and dephosphorylation of a 62,000 relative molecular weight (Mr) protein in autoradiograms of sodium dodecyl sulfate polyacrylamide gels originating from canine renal brush border membranes. In the current studies 32P phosphorylation of the 62,000 Mr protein that was independent of cAMP was noted in the presence of Zn2+. Under these conditions, cAMP inhibited the 32P phosphorylation of this protein. Concentration-dependent photoaffinity labeling of a band with Mr 60,000 in autoradiograms of gels resulted from incubation of membranes with cyclic 8-azidoadenosine-3',5'-monophosphate (8-N3-[32P]cAMP) followed by exposure to light. In the presence of Zn2+ and ATP, an apparent shift in the Mr of a portion of the photoaffinity-labeled band to 62,000 was seen. The 62,000 Mr phosphoprotein in detergent-solubilized supernatants of brush border membranes was immunoprecipitated with antibodies directed against the regulatory subunit of type II cAMP-dependent protein kinase. Our observations strongly suggest that the 62,000 Mr protein is the regulatory subunit.

Animals↗