Calcium transients in human platelets monitored by aequorin, fura-2 and quin-2: effects of protein kinase C activation and inhibition

Tumour-promoting phorbol esters and 1,2-dioctanoyl-sn-glycerol both induce calcium transients in platelets. However, these can only be detected in platelets loaded with aequorin, but not in those loaded with the fluorescent probes quin-2 and fura-2 presumably because of intracellular calcium buffering. Several effects induced by phorbol esters and diacylglycerols, including the rise in (Ca2+)i, the stimulation of Na+/H+ transporter and the inhibition of the effects of thrombin alone on (Ca2+)i are potently antagonised by staurosporine, a compound known to inhibit protein kinase C. Higher concentrations of staurosporine themselves inhibit the thrombin-induced calcium transient. Staurosporine inhibits the effects of phorbol esters and dioctanoyl glycerol with equal potency although the latter does not cause enzyme translocation of cytosolic protein kinase C to membranes. These results therefore suggest that some, if not all, the effects of protein kinase C activation can occur without translocation of the enzyme.     

Cytoskeletal protein and mRNA accumulation during brush border formation in adult chicken enterocytes

We have explored the development of the brush border in adult chicken enterocytes by analyzing the cytoskeletal protein and mRNA levels as enterocytes arise from crypt stem cells and differentiate as they move toward the villus. At the base of the crypt, a small population of cells contain a rudimentary terminal web and a few short microvilli with long rootlets. These microvilli appear to arise from bundles of actin filaments which nucleate on the plasma membrane. The microvilli apparently elongate via the addition of membrane supplied by vesicles that fuse with the microvillus and extend the membrane around the actin core. Actin, villin, myosin, tropomyosin and spectrin, but not myosin I (previously called 110 kD; see Mooseker and Coleman, J. Cell Biol. 108, 2395-2400, 1989) are already concentrated in the luminal cytoplasm of crypt cells, as seen by immunofluorescence. Using quantitative densitometry of cDNA-hybridized RNA blots from cells isolated from crypts, villus middle (mid), or villus tip (tip), we found a 2- to 3-fold increase in villin, calmodulin and tropomyosin steady-state mRNA levels; an increase parallel to morphological brush border development. Actin, spectrin and myosin mRNA levels did not change significantly. ELISA of total crypt, mid and tip cell lysates show that there are no significant changes in actin, myosin, spectrin, tropomyosin, myosin I, villin or alpha-actinin protein levels as the brush border develops. The G-/F-actin ratio also did not change with brush border assembly. We conclude that, although the brush border is not fully assembled in immature enterocytes, the major cytoskeletal proteins are present in their full concentration and already localized within the apical cytoplasm. Therefore brush border formation may involve reorganization of a pool of existing cytoskeletal proteins mediated by the expression or regulation of an unidentified key protein(s).     

Kinetic analysis of L-glutamine transport into porcine jejunal enterocyte brush-border membrane vesicles

L-Glutamine transport into porcine jejunal enterocyte brush border membrane vesicles was studied. Uptake was mediated by a Na(+)-dependent and a Na(+)-independent pathway as well as by diffusion. The initial rates of glutamine uptake over a range of concentrations is both Na(+)-gradient and Na(+)-free conditions were analyzed and kinetic parameters were obtained. Na(+)-dependent glutamine transport had a K(m) of 0.77 +/- 0.16 mM and a Jmax of 70.7 +/- 5.8 pmol mg protein-1 s-1; Na(+)-independent glutamine transport had a K(m) of 3.55 +/- 0.78 mM and a Jmax of 55.1 +/- 6.6 pmol mg protein-1 s-1. The non-saturable component measured with HgCl2-poisoned brush border membrane vesicles in the Na(+)-free condition contained passive diffusion and non-specific membrane binding and was defined to be apparent glutamine diffusion and the glutamine permeability coefficient (Kdiff) was estimated to be Kdiff = 3.78 +/- 0.06 pmol 1 mg protein-1 mmol-1 s-1. Results of inhibition experiments showed that Na(+)-dependent glutamine uptake occurred primarily through the brush border system-B degree transporters, whereas Na(+)-independent glutamine uptake occurred via the system-L transporters. Furthermore, the kinetics of L-leucine and L-cysteine inhibition of L-glutamine uptake demonstrated that neutral amino acids sharing the same brush border transporters can effectively inhibit each other in their transport.     

Streptozotocin diabetes and sugar transport by rat ileal enterocytes: evidence for adaptation caused by an increased luminal nutrient load.

Preparations of villus enterocytes and brush border membrane vesicles have been used to study the effects of streptozotocin-induced diabetes mellitus in rats on sugar transport across the brush border and basolateral membranes of ileal epithelial cells. In isolated cells, diabetes increased Na(+)-dependent galactose transport across the brush border of mid-villus but not upper villus cells. Galactose transport across the basolateral membrane was, however, enhanced by diabetes in both cell populations. Kinetic analysis of vesicle data suggested the presence of two transporters for Na(+)-dependent glucose transport. Diabetes induced a 5-fold increase in both KT and Vmax of the high-affinity/low-capacity system together with a 2-fold increase in the Vmax of the low-affinity/high-capacity transporter. Glucose was almost undetectable in the lumen of the upper and lower ileum in control animals but was present at high levels (26.1 +/- 4.3 mM and 6.5 +/- 1.3 mM) in diabetic rats. The possible significance of these changes in luminal sugar concentration in relation to the adaptation of transport across ileal enterocytes is discussed.     

Intestinal calcium-binding protein

Summary The vitamin D-dependent calcium-binding protein (CaBP) was studied in relation to the age of the cell, in isolated epithelial cell populations removed from rat duodenum. Alkaline phosphatase and thymidine kinase activities were used as markers to characterize differentiated villus cells and undifferentiated (mitotically active) crypt cells, respectively. CaBP distribution along the length of the villus, as established by radioimmunoassay, appears as a gradient increasing from the crypt to the tip of the villus. CaBP concentration in cells is shown to be (i) negatively correlated with the thymidine kinase activity of cells, and (ii) positively correlated with the alkaline phosphatase activity of cells. This indicates that CaBP is absent in crypt cells and appears in differentiated cells with the development of the brush border. Thus CaBP, like alkaline phosphatase, can be considered as an indicator of enterocyte maturation. These data were also confirmed by studying the cellular localization of the protein. In addition both indirect immunofluorescence and immunoperoxidase staining methods reveal that antibody against CaBP decorates the terminal web, but not the microvilli of the brush border of mature absorptive cells. The results suggest that CaBP may act as a modulator of some Ca²⁺-mediated biochemical processes at the level of the enterocyte brush border.Portions of this work were presented at the Fourth International Workshop on Calcified Tissues, Israel (March 1980)     

Aminopeptidase N (CD13) is a molecular target of the cholesterol absorption inhibitor ezetimibe in the enterocyte brush border membrane

Intestinal cholesterol absorption is an important regulator of serum cholesterol levels. Ezetimibe is a specific inhibitor of intestinal cholesterol absorption recently introduced into medical practice; its mechanism of action, however, is still unknown. Ezetimibe neither influences the release of cholesterol from mixed micelles in the gut lumen nor the transfer of cholesterol to the enterocyte brush border membrane. With membrane-impermeable Ezetimibe analogues we could demonstrate that binding of cholesterol absorption inhibitors to the brush border membrane of small intestinal enterocytes from the gut lumen is sufficient for inhibition of cholesterol absorption. A 145-kDa integral membrane protein was identified as the molecular target for cholesterol absorption inhibitors in the enterocyte brush border membrane by photoaffinity labeling with photoreactive Ezetimibe analogues (Kramer, W., Glombik, H., Petry, S., Heuer, H., Schafer, H. L., Wendler, W., Corsiero, D., Girbig, F., and Weyland, C. (2000) FEBS Lett. 487, 293-297). The 145-kDa Ezetimibe-binding protein was purified by three different methods and sequencing revealed its identity with the membrane-bound ectoenzyme aminopeptidase N ((alanyl)aminopeptidase; EC 3.4.11.2; APN; leukemia antigen CD13). The enzymatic activity of APN was not influenced by Ezetimibe (analogues). The uptake of cholesterol delivered by mixed micelles by confluent CaCo-2 cells was partially inhibited by Ezetimibe and nonabsorbable Ezetimibe analogues. Preincubation of confluent CaCo-2 cells with Ezetimibe led to a strong decrease of fluorescent APN staining with a monoclonal antibody in the plasma membrane. Independent on its enzymatic activity, aminopeptidase N is involved in endocytotic processes like the uptake of viruses. Our findings suggest that binding of Ezetimibe to APN from the lumen of the small intestine blocks endocytosis of cholesterol-rich membrane microdomains, thereby limiting intestinal cholesterol absorption.     

Mechanism of regulation of rabbit intestinal villus cell brush border membrane Na/H exchange by nitric oxide

In the mammalian small intestine, coupled NaCl absorption occurs via the dual operation of Na/H and Cl/HCO(3) exchange on the villus cell brush border membrane (BBM). Although constitutive nitric oxide (cNO) has been demonstrated to alter gastrointestinal tract functions, how cNO may specifically alter these two transporters to regulate coupled NaCl absorption is unknown. In villus cells, inhibition of cNO synthase (cNOS) with l-N(G)-nitroarginine methylester (l-NAME) stimulated Na/H exchange whereas Cl/HCO(3) exchange was unaffected. In villus cell BBM vesicles (BBMV) prepared from rabbits treated with l-NAME, Na/H exchange was also stimulated. d-NAME, an inactive analog of l-NAME, and N(6)-(1-imonoethyl)-l-lysine dihydrochloride, a more selective inhibitor of inducible NO synthase, did not affect Na/H exchange. Kinetic studies demonstrated that the mechanism of stimulation is secondary to an increase in the maximal rate of uptake of Na, without an alteration in the affinity of the transporter for Na. Northern blot studies demonstrated an increase in the message for the BBM Na/H exchanger NHE3, and Western blot studies showed that the immunoreactive protein levels of NHE3 was increased when cNOS was inhibited. Thus these results indicate that cNO under nominal physiological states most likely maintains an inhibitory tone on small intestinal coupled NaCl absorption by specifically inhibiting BBM Na/H expression.     

Interaction of renin inhibitors with the intestinal uptake system for oligopeptides and beta-lactam antibiotics

The interaction of two renin inhibitors, S 86,2033 and S 86,3390, with the uptake system for beta-lactam antibiotics and small peptides in the brush border membrane of enterocytes from rabbit small intestine was investigated using brush border membrane vesicles. Both renin inhibitors inhibited the uptake of the orally active cephalosporin cephalexin into brush border membrane vesicles from rabbit small intestine in a concentration-dependent manner. 1.1 mM of S 86,3390 and 2.5 mM of S 86,2033 led to a half-maximal inhibition of the H(+)-dependent uptake of cephalexin. Both renin inhibitors were stable against peptidases of the brush border membrane. The uptake of cephalexin into brush border membrane vesicles (1 min of incubation) was competitively inhibited by S 86,2033 and S 86,3390 suggesting a direct interaction of these compounds with the intestinal peptide uptake system. The renin inhibitors are transported across the brush border membrane into the intravesicular space as was shown by equilibrium uptake studies dependent upon the medium osmolarity. The uptake of S 86,3390 was stimulated by an inwardly directed H(+)-gradient and occurred with a transient accumulation against a concentration gradient (overshoot phenomenon). The renin inhibitors S 86,2033 and 86,3390 also caused a concentration-dependent inhibition in the extent of photoaffinity labeling of the putative peptide transport protein of apparent Mr 127,000 in the brush border membrane of small intestinal enterocytes. In conclusion, these studies show that renin inhibitors specifically interact with the intestinal uptake system shared by small peptides and beta-lactam antibiotics.     

Thyroid hormones stimulate calcium transport systems in rat intestine

Thyroid hormone status influences calcium metabolism. To elucidate the mechanism of action of thyroid hormones on transcellular transport of calcium in rat intestine, Ca(2+) influx and efflux studies were carried out in brush border membrane vesicles (BBMV) and across the basolateral membrane (BLM) of enterocytes, respectively. Steady-state uptake of Ca(2+) into BBMV as well as Ca(2+) efflux from the BLM enterocytes was significantly increased in hyperthyroid (Hyper-T) rats and decreased in hypothyroid (Hypo-T) rats as compared to euthyroid (Eu-T) rats. Kinetic studies revealed that increase in steady state Ca(2+) uptake into BBMV from hyper-T rats was fraternized with decrease in Michaelis Menten Constant (K(m)), indicating a conformational change in Ca(2+) transporter. Further, this finding was supported by significant changes in transition temperature and membrane fluidity. Increased Ca(2+) efflux across enterocytes was attributed to sodium-dependent Ca(2+) exchange activity which was significantly higher in Hyper-T rats and lower in Hypo-T rats as compared to Eu-T rats. However, there was no change in Ca(2+)-ATPase activity of BLMs of all groups. Kinetic studies of Na(+)/Ca(2+) exchanger revealed that alteration in Na(+)-dependent Ca(2+) efflux was directly associated with maximal velocity (V(max)) of exchanger among all the groups. cAMP, a potent activator of Na(+)/Ca(2+) exchanger, was found to be significantly higher in intestinal mucosa of Hyper-T rats as compared to Eu-T rats. Therefore, the results of this study suggest that Ca(2+) influx across BBM is possibly modulated by thyroid hormones by mediating changes in membrane fluidity. Thyroid hormones activated the Na(+)/Ca(2+) exchange in enterocytes possibly via cAMP-mediated pathway.     

Intestinal uptake of dipeptides and beta-lactam antibiotics. I. The intestinal uptake system for dipeptides and beta-lactam antibiotics is not part of a brush border membrane peptidase

The uptake of beta-lactam antibiotics into small intestinal enterocytes occurs by the transport system for small peptides. The role of membrane-bound peptidases in the brush border membrane of enterocytes from rabbit and pig small intestine for the uptake of small peptides and beta-lactam antibiotics was investigated using brush border membrane vesicles. The enzymatic activity of aminopeptidase N was inhibited by beta-lactam antibiotics in a non-competitive manner whereas dipeptidylpeptidase IV was not affected. The peptidase inhibitor bestatin led to a strong competitive inhibition of aminopeptidase N whereas the uptake of cephalexin into brush border membrane vesicles was only slightly inhibited at high bestatin concentrations (greater than 1 mM). Modification of brush border membrane vesicles with the histidine-modifying reagent diethyl pyrocarbonate led to a strong irreversible inhibition of cephalexin uptake whereas the activity of aminopeptidase N remained unchanged. A modification of serine residues with diisopropyl fluorophosphate completely inactivated dipeptidylpeptidase IV whereas the transport activity for cephalexin and the enzymatic activity of aminopeptidase N were not influenced. With polyclonal antibodies raised against aminopeptidase N from pig renal microsomes the aminopeptidase N from solubilized brush border membranes from pig small intestine could be completely precipitated; the binding protein for beta-lactam antibiotics and oligopeptides of apparent Mr 127,000 identified by direct photoaffinity labeling with [3H]benzylpenicillin showed no crossreactivity with the aminopeptidase N anti serum and was not precipitated by the anti serum. These results clearly demonstrate that peptidases of the brush border membrane like aminopeptidase N and dipeptidylpeptidase IV are not directly involved in the intestinal uptake process for small peptides and beta-lactam antibiotics and are not a constituent of this transport system. This suggests that a membrane protein of Mr 127,000 is (a part of) the uptake system for beta-lactam antibiotics and small peptides in the brush border membrane of small intestinal enterocytes.     

OCTN3: A Na+-independent L-carnitine transporter in enterocytes basolateral membrane

L-carnitine transport has been measured in enterocytes and basolateral membrane vesicles (BLMV) isolated from chicken intestinal epithelia. In the nominally Na+-free conditions chicken enterocytes take up L-carnitine until the cell to medium L-carnitine ratio is 1. This uptake was inhibited by L-carnitine, D-carnitine, gamma-butyrobetaine, acetylcarnitine, tetraethylammonium (TEA), and betaine. L-3H-carnitine uptake into BLMV showed no overshoot, and it was (i) Na+-independent, (ii) trans-stimulated by intravesicular L-carnitine, and (iii) cis-inhibited by TEA and cold L-carnitine. L-3H-carnitine efflux from L-3H-carnitine preloaded enterocytes was also Na+-independent, and trans-stimulated by L-carnitine, D-carnitine, gamma-butyrobetaine, acetylcarnitine, TEA, and betaine. Both, uptake and efflux of L-carnitine were inhibited by verapamil and unaffected by either extracellular pH or palmitoyl-L-carnitine. RT-PCR with specific primers for the mouse OCTN3 transporter revealed the existence of OCTN3 mRNA in mouse intestine, which was confirmed by in situ hybridization studies. Immunohystochemical analysis showed that OCTN3 protein was mainly associated with the basolateral membrane of rat and chicken enterocytes, whereas OCTN2 was detected at the apical membrane. In conclusion, the results demonstrate for the first time that (i) mammalian small intestine expresses OCTN3 mRNA along the villus and (ii) that OCTN3 protein is located in the basolateral membrane. They also suggest that OCTN3 could mediate the passive, Na+ and pH-independent L-carnitine transport activity measured in the three experimental conditions.     

Na+-coupled D-glucose uptake and membrane order of enterocyte brush border membrane vesicles, under the effect of a series of N-phenylcarbamates

The importance of the hydrophobic effect of exogenous substances and of modifications of membrane order on D-glucose uptake are still poorly defined. Our results show that the concentrative Na+ -coupled D-glucose uptake of rat enterocyte brush border membrane vesicles is inhibited by N-phenylcarbamates increase the membrane order. However, since the concentrations required for membrane order increase are much greater than those active on D-glucose uptake, the effects on lipid order cannot be responsible for the inhibition of D-glucose uptake. Measurements of D-glucose uptake under conditions of Na+ equilibrium show that these carbamates do not act directly on the carrier but indirectly by favouring the dissipation of the Na+ gradient.     

Rap2A links intestinal cell polarity to brush border formation

The microvillus brush border at the apex of the highly polarized enterocyte allows the regulated uptake of nutrients from the intestinal lumen. Here, we identify the small G protein Rap2A as a molecular link that couples the formation of microvilli directly to the preceding cell polarization. Establishment of apicobasal polarity, which can be triggered by the kinase LKB1 in single, isolated colon cells, results in enrichment of PtdIns(4,5)P(2) at the apical membrane. The subsequent recruitment of phospholipase D1 allows polarized accumulation of phosphatidic acid, which provides a local cue for successive signalling by the guanine nucleotide exchange factor PDZGEF, the small G protein Rap2A, its effector TNIK, the kinase MST4 and, ultimately, the actin-binding protein Ezrin. Thus, epithelial cell polarization is translated directly into the acquisition of brush borders through a small G protein signalling module whose action is positioned by a cortical lipid cue.     

Nitric oxide inhibits enterocyte migration through activation of RhoA-GTPase in a SHP-2-dependent manner

Diseases of intestinal inflammation like necrotizing enterocolitis (NEC) are associated with impaired epithelial barrier integrity and the sustained release of intestinal nitric oxide (NO). NO modifies the cytoskeletal regulator RhoA-GTPase, suggesting that NO could affect barrier healing by inhibiting intestinal restitution. We now hypothesize that NO inhibits enterocyte migration through RhoA-GTPase and sought to determine the pathways involved. The induction of NEC was associated with increased enterocyte NO release and impaired migration of bromodeoxyuridine-labeled enterocytes from terminal ileal crypts to villus tips. In IEC-6 enterocytes, NO significantly inhibited enterocyte migration and activated RhoA-GTPase while increasing the formation of stress fibers. In parallel, exposure of IEC-6 cells to NO increased the phosphorylation of focal adhesion kinase (pFAK) and caused a striking increase in cell-matrix adhesiveness, suggesting a mechanism by which NO could impair enterocyte migration. NEC was associated with increased expression of pFAK in the terminal ileal mucosa of wild-type mice and a corresponding increase in disease severity compared with inducible NO synthase knockout mice, confirming the dependence of NO for FAK phosphorylation in vivo and its role in the pathogenesis of NEC. Strikingly, inhibition of the protein tyrosine phosphatase SHP-2 in IEC-6 cells prevented the activation of RhoA by NO, restored focal adhesions, and reversed the inhibitory effects of NO on enterocyte migration. These data indicate that NO impairs mucosal healing by inhibiting enterocyte migration through activation of RhoA in a SHP-2-dependent manner and support a possible role for SHP-2 as a therapeutic target in diseases of intestinal inflammation like NEC.     

Dual effect of cyclic GMP on renal brush border Na-H antiporter.

The Na-H antiporter of renal-brush border membranes is inhibited by cyclic AMP and stimulated by protein kinase C. The proximal tubule contains guanylate cyclase and is capable of cyclic GMP production. The effect of cGMP on renal Na-H antiporter activity was analyzed in phosphorylated brush border membranes by 22Na uptake in the presence or absence of 1 mM amiloride. 8-Bromo cyclic GMP (1 microM) increased the amiloride-sensitive 22Na uptake in control from 1.26 +/- 0.13 to 1.54 +/- 0.12 nmol/mg/protein/10 sec, P less than 0.01, without altering the amiloride-insensitive component. In the absence of exogenous ATP, cGMP also stimulated the amiloride-sensitive 22Na uptake, which can be explained by the presence of endogenous ATP in concentrations of up to 50 microM in the membranes. In ATP-depleted membrane vesicles, however, cGMP inhibited the amiloride-sensitive 22Na uptake. These data indicate that cGMP acts on the Na-H antiporter by at least two different mechanisms, one of which is ATP dependent. It is likely that cGMP-dependent protein kinase mediates the stimulatory effects seen in the presence of ATP, and the inhibition seen in ATP-depleted membranes results from cGMP direct action on the Na-H antiporter.     

The administration of lipopolysaccharide, in vivo, induces alteration in L-leucine intestinal absorption

The objective of the present study was to determine the alterations in L-leucine intestinal uptake by intravenous administration of Lipopolysaccharide (LPS), which is a constituent of gram negative bacterial, causative agent of sepsis. The amino acid absorption in LPS treated rabbits was reduced compared to the control animals. The LPS effect on the amino acid uptake was due to an inhibition of the Na+-dependent system of transport, through both reduction of the apparent capacity transport (Vmax) and diminution of the Na+/K-ATPase activity. The results have also shown that the LPS decreases the mucosal to serosal transepithelial flux and the transport across brush border membrane vesicles of L-leucine. The study of possible intracellular mechanisms implicated in the LPS effect, showed that the second messengers calcium, protein kinase C and c-AMP did not play any role in this effect. However, the absence of ion chloride in the incubation medium removes the LPS inhibition and the intracellular tissue water was affected by the LPS treatment. Therefore, the inhibition in the L-leucine intestinal absorption, by intravenous administration of LPS, could be mainly produced by the secretagogue action of this endotoxin on the gut.     

Protein kinase C in mouse kidney: subcellular distribution and endogenous substrates

The subcellular distribution, kinetic properties, and endogenous substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) were examined in mouse kidney cortex. Protein kinase C associated with the particulate, mitochondrial, and brush border membrane fractions was assayed after solubilization in 0.2% Triton X-100 under conditions shown to be noninhibitory to catalytic activity. Of recovered activity, 52% was associated with the cytosolic fraction; mitochondrial and brush border membrane associated protein kinase C constituted 12 and 3%, respectively, of the activity recovered in the particulate fraction. Protein kinase C associated with brush border membranes exhibited a high affinity for ATP (apparent Km = 62 +/- 10 microM) and the highest apparent maximal velocity (1146 +/- 116 pmol P/(mg protein.min] of the renal fractions examined. Maximal stimulation of protein kinase C by diacylglycerol (in the presence of phosphatidylserine) was achieved at both 25 and 300 microM calcium in all renal fractions. These results are consistent with previous reports demonstrating that diacylglycerol increases the apparent affinity of protein kinase C for calcium. Phorbol 12-myristate 13-acetate, but not 4 alpha-phorbol, was able to substitute for diacylglycerol and stimulate cytosolic and particulate renal protein kinase C. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a specific inhibitor of protein kinase C, led to significant inhibition of catalytic activity in all renal subcellular fractions. Endogenous substrates for protein kinase C were demonstrated in renal cytosolic (26, 45, 63, and 105 kilodaltons (kDa], particulate (26, 33, 68, and 105 kDa), mitochondrial (43 kDa), and brush border membrane (26, 41, 52, 88, and 105 kDa) fractions. The possible physiological significance of protein kinase C in mammalian kidney is discussed.     

Pharmacological investigations on the lipolytic and antilipolytic effects of growth hormone (GH) in chicken adipose tissue in vitro: evidence for involvement of calcium and polyamines

The involvement of RNA/protein synthesis, calcium, calmodulin, protein kinase C, and polyamines in the lipolytic and antilipolytic (inhibition of glucagon-stimulated lipolysis) responses to GH have been investigated employing chicken adipose tissue in vitro. The lipolytic, but not the antilipolytic, effect of GH was blocked by inhibitors of RNA/protein synthesis (actinomycin D, cycloheximide, and puromycin) and calcium uptake (verapamil) and low calcium concentrations (0.28 mM CaCl2). The antilipolytic, but not the lipolytic, effect of GH was blocked by alpha-difluoromethylornithine (DFMO), a polyamine synthesis inhibitor. DFMO-induced blockade of the antilipolytic GH response was reversed by the addition of spermidine. The lipolytic and antilipolytic effects of GH were not influenced by chlorpromazine (a calmodulin inhibitor) or phorbol 12-myristate 13-acetate (PMA) (an activator of protein kinase C).     

Phosphorylation of brush border myosin I by protein kinase C is regulated by Ca(2+)-stimulated binding of myosin I to phosphatidylserine concerted with calmodulin dissociation.

Brush border myosin I from chicken intestine is phosphorylated in vitro by chicken intestinal epithelial cell protein kinase C. Phosphorylation on serine and threonine to a maximum of 0.93 mol of P/mol of myosin I occurs within an approximately 20 kDa region at the end of the COOH-terminal tail of the 119-kDa heavy chain. The effects of Ca2+ on myosin I phosphorylation by protein kinase C are complex, with up to 4-fold stimulation occurring at 0.5-3 microM Ca2+, and up to 80% inhibition occurring at 3-320 microM Ca2+. Phosphorylation required that brush border myosin I be in its phosphatidylserine vesicle-bound state. Previously unknown Ca2+ stimulation of brush border myosin I binding to phosphatidylserine vesicles was found to coincide with Ca2+ stimulation of phosphorylation. A myosin I proteolytic fragment lacking approximately 20 kDa of its tail retained Ca(2+)-stimulated binding, but showed reduced Ca(2+)-independent binding. Ca(2+)-dependent phosphatidylserine binding is apparently due to the concomitant phosphatidylserine-promoted, Ca(2+)-induced dissociation of up to three of the four calmodulin light chains from myosin I. Four highly basic putative calmodulin-binding sites in the Ca(2+)-dependent phosphatidylserine binding region of the heavy chain were identified based on the similarity in their sequence to the calmodulin- and phosphatidylserine-binding site of neuromodulin. Calmodulin dissociation is now shown to occur in the low micromolar Ca2+ concentration range and may regulate the association of brush border myosin I with membranes and its phosphorylation by protein kinase C.