Pharmacological Characterization of a Novel Beta 3 Adrenergic Agonist, Vibegron: Evaluation of Antimuscarinic Receptor Selectivity for Combination Therapy for Overactive Bladder.

Although the physiologic role of muscarinic receptors in bladder function and the therapeutic efficacy of muscarinic antagonists for the treatment of overactive bladder are well established, the role of ß3-adrenergic receptors (ß3ARs) and their potential as therapeutics is just emerging. In this manuscript, we characterized the pharmacology of a novel ß3AR agonist vibegron (MK-4618, KRP-114V) and explored mechanistic interactions of ß3AR agonism and muscarinic antagonism in urinary bladder function. Vibegron is a potent, selective full ß3AR agonist across species, and it dose dependently increased bladder capacity, decreased micturition pressure, and increased bladder compliance in rhesus monkeys. The relaxation effect of vibegron was enhanced when combined with muscarinic antagonists, but differentially influenced by muscarinic receptor subtype selectivity. The effect was greater when vibegron was co-administered with tolterodine, a nonselective antagonist, compared with coadministration with darifenacin, a selective M3 antagonist. Furthermore, a synergistic effect for bladder strip relaxation was observed with the combination of a ß3AR agonist and tolterodine in contrast to simple additivity with darifenacin. To determine expression in rhesus bladder, we employed a novel ß3AR agonist probe, [3H]MRL-037, that selectively labels ß3 receptors in both urothelium and detrusor smooth muscle. Vibegron administration caused a dose-dependent increase in circulating glycerol and fatty acid levels in rhesus and rat in vivo, suggesting these circulating lipids can be surrogate biomarkers. The translation of our observation to the clinic has yet to be determined, but the combination of ß3AR agonists with M2/M3 antimuscarinics has the potential to redefine the standard of care for the pharmacological treatment of overactive bladder.

SPECTRON, a neutron noise measurement system in frequency domain.

This paper is dedicated to the presentation and validation of SPECTRON, a novel neutron noise measurement system developed at CEA Cadarache. The device is designed for the measurement of the ß(eff) parameter (effective fraction of delayed neutrons) of experimental nuclear reactors using the Cohn-α method. An integrated electronic system is used to record the current from fission chambers. Spectra computed from measurement data are processed by a dedicated software in order to estimate the reactor transfer function and then the effective fraction of delayed neutrons as well as the prompt neutron generation time. After a review of the pile noise measurement method in current mode, the SPECTRON architecture is presented. Then, the validation procedure is described and experimental results are shown, supporting the proper functioning of this new measurement system. It is shown that every technical requirement needed for correct measurement of neutron noise is fulfilled. Measurements performed at MINERVE and EOLE, two experimental nuclear reactors at CEA Cadarache, in real conditions allowed us to validate SPECTRON.

Cyclic imides as potent and selective alpha-1A adrenergic receptor antagonists.

We disclose a new compound class of potent and selective alpha-1A adrenergic receptor antagonists exemplified by the geminally, disubstituted cyclic imide 7. The optimization of lead compounds resulting in the cyclic imide motif is highlighted. The results of in vitro and in vivo studies of selected compounds are presented.

The CXCR4 agonist ligand stromal derived factor-1 maintains high affinity for receptors in both Galpha(i)-coupled and uncoupled states.

The alpha chemokine receptor CXCR4 and its only characterized chemokine ligand, stromal cell-derived factor-1 (SDF-1), are postulated to be important in the development of the B-cell arm of the immune system. In addition, CXCR4 is a critical coreceptor in support of viral entry by T-cell line tropic strains (X4) of the Human Immunodeficiency Virus Type 1 (HIV-1), viral variants which predominate in some infected individuals in end stage disease. SDF-1 can block X4-tropic HIV-1 infection of CD4+ target cells in vitro, and allelic variants of the human gene encoding SDF-1 in vivo correlate with delayed disease progression. Therefore, CXCR4 may be an appropriate target for therapeutic intervention in acquired immunodeficiency syndrome (AIDS), and knowledge of the pharmacology of SDF-1 binding to its cognate receptor will be important in the interpretation of these experiments. We report here a Kd derived using a competition binding assay of 4.5 nM for CXCR4 endogenously expressed on peripheral blood monocytes and T-cells. This affinity is similar to that which SDF-1 exhibits when binding to endogenous CXCR4 on an established immortal Jurkat T-cell line as well as recombinant CXCR4 transfected into Chinese Hamster Ovary (CHO) cells. We also demonstrate that the determined affinity of SDF-1 for CXCR4 is reflective of its ability to induce a CXCR4-mediated signal transduction in these different cell types. Furthermore, using Bordetella pertussis toxin, we observe that high affinity binding of SDF-1 to CXCR4 is independent of the G-protein coupled state of the receptor, as uncoupling of G-protein did not lead to the appearance of measurable low affinity SDF-1 binding sites. Moreover, binding affinity and receptor number were unaffected by uncoupling for both recombinant and endogenously expressed CXCR4. Thus, SDF-1 is novel among agonist ligands of G protein-coupled receptors in that it appears to have equal affinity for both the G protein-coupled and uncoupled states of CXCR4.

Phenylacetamides as selective alpha-1A adrenergic receptor antagonists.

A novel class of potent and selective alpha-1a receptor antagonists has been identified. The structures of these antagonists were derived from truncating the 4-aryl dihydropyridine subunit present in known alpha-1a antagonists. The design principles which led to the discovery of substituted phenylacetamides, the synthesis and SAR of key analogues, and the results of select in vitro and in vivo studies are described.

Acidic fibroblast growth factor is present in the enteric nervous system of the large intestine.

Acidic fibroblast growth factor (aFGF) is a heparin binding protein that displays pleiotropic activity. The purpose of this study was to document the presence of the translated aFGF product, its mRNA, and its location in the colon. mRNA was extracted from bovine large intestine and reverse transcribed to cDNA. Nested-primer PCR was used to determine the presence of mRNA using primers homologous to the previously published bovine aFGF cDNA. Purification of translated aFGF was performed using an established HPLC protocol. Western blot analysis of the HPLC fractions was performed using two epitope-independent antibodies against aFGF. Immunohistochemistry employed these antibodies to determine the locus of aFGF expression. The nested-primer PCR product of predicted size was homologous to the published bovine aFGF mRNA sequence, as determined by DNA sequencing. Intestinal aFGF had a mass similar to bovine aFGF isolated from other tissues, and immunocrossreacted with two peptide-based, epitope-independent anti-aFGF antisera on Western blotting. Immunohistochemical analysis of large intestine using these two independent antisera localized aFGF within the myenteric plexus. These data demonstrate that aFGF is present within the myenteric plexus of the enteric nervous system.

Protein kinase C: modulation of vasopressin-induced calcium influx and release in A7r5 vascular smooth muscle cells.

This study was guided by the hypothesis that specific isoforms of protein kinase C may participate in modulating increases in intracellular Ca2+ that are induced by stimulation of vascular smooth muscle cells with vasopressin. Immunoblot analysis revealed that A7r5 vascular smooth muscle cells expressed conventional (alpha), novel (delta and epsilon), and atypical (iota/lambda and mu) isoforms of protein kinase C. Stimulation of fura-2-loaded cells with 20 nM vasopressin induced a rapid transient increase in the intracellular concentration of calcium that was followed by a slowly declining component which was above baseline throughout the period of observation. Cell fractionation studies showed that the calcium response was associated with (a) transient translocation of the alpha and delta isoforms of protein kinase C from the cytosolic fraction to the particulate-membrane fraction, (b) sustained translocation of the epsilon isoform, and (c) no translocation of iota/lambda or mu isoforms. Ratiometric and isobestic fluorescence analysis showed that vasopressin-induced Ca2+ influx and release were markedly inhibited in cells that were preincubated with either 1 microM phorbol 12-myristate 13-acetate, or 10 microM 1, 2 dioctanoyl-sn-glycerol, two structurally different activators of protein kinase C. In contrast, vasopressin-induced increases in intracellular Ca2+ were not significantly altered following preincubation with either 1 microM 4alpha-phorbol or 4alpha-phorbol 12,13-didecanoate, analogs of phorbol 12-myristate 13-acetate that do not activate protein kinase C. Moreover, the inhibitory effects of phorbol 12-myristate 13-acetate were prevented by treatment with 1 microM GF109203X, a potent inhibitor of protein kinase C. Taken together, these results show that direct activation of protein kinase C can negatively modulate vasopressin-induced Ca2+ influx and release in cultured vascular smooth muscle cells. They also show that stimulation with vasopressin induces translocation of specific isoforms of protein kinase C, an observation suggesting that one or more of these isoforms may participate in modulation of vasopressin-induced increases in intracellular Ca2+.

Stimulation of G-protein coupled receptors in vascular smooth muscle cells induces tyrosine kinase dependent increases in calcium without tyrosine phosphorylation of phospholipase C gamma-1.

It is often believed that increases in intracellular Ca2+ ([Ca2+]i) resulting from stimulation of G-protein coupled receptors in vascular smooth muscle cells (VSMC) require activation of the beta1 isoform of phospholipase C (PLC). However, recent studies showed that rat aortic VSMC do not express PLC beta-1 and that stimulation with angiotensin-II induces tyrosine kinase dependent increases in [Ca2+]i and tyrosine phosphorylation of PLC gamma-1. Whether this pathway is activated by other vasoactive agents that stimulate G-protein coupled receptors is unknown. Here, we show that A10 VSMC express PLC beta-2, PLC beta-3, PLC delta-1, and PLC gamma-1. The cells also expressed Galpha(q/11). However, neither PLC beta-1 nor PLC beta-4 was detected. Stimulation with angiotensin-II, vasopressin, serotonin, or endothelin induced tyrosine kinase dependent increases in [Ca2+]i. However, tyrosine phosphorylation of PLC gamma-1 did not occur. In contrast, stimulation with platelet derived growth factor increased [Ca2+]i and tyrosine phosphorylation of PLC gamma-1. The results show that tyrosine phosphorylation of PLC gamma-1 is not required for tyrosine kinase dependent increases in [Ca2+]i resulting from stimulation of diverse G-protein coupled receptors in VSMC.

Selective alpha-1a adrenergic receptor antagonists. Effects of pharmacophore regio- and stereochemistry on potency and selectivity.

The anti-anxiety agent ipsapirone has been shown to have modest affinity for alpha-1 receptors. We disclose the discovery of potent alpha-1a receptor subtype selective antagonists based on the ipsapirone structure which possess selectivity versus the 5-HT receptors tested. These antagonists were obtained by tethering a saccharin ring to 4-phenyl-3-carboxyethyl piperidines. The design principles which led to this structural motif are discussed. The synthesis of key analogs, their SAR, as well as results of selected in vitro and in vivo studies are described.

Genistein sensitivity of calcium transport pathways in serotonin-activated vascular smooth muscle cells.

Recent studies showed that serotonin-activated increases in intracellular Ca2+ in vascular smooth muscle cells are associated with enhanced protein tyrosine phosphorylation. These responses were blocked by inhibition of tyrosine kinase activity with genistein, suggesting that the increases in Ca2+ and tyrosine phosphorylation are functionally coupled. Therefore, we sought to characterize genistein-sensitive Ca2+ transport pathways in rat aortic A10 cells loaded with fura-2. In the presence of extracellular Ca2+, serotonin evoked a transient increase in [Ca2+]i that was followed by a smaller sustained increase. The transient was inhibited 25-40% by L-type Ca2+ channel antagonists and inhibited 90-95% by genistein. The sustained response was unaffected by L-channel antagonists and only slightly inhibited by genistein. In the absence of extracellular Ca2+, the transient was reduced by 50%, while the sustained component was virtually abolished. These results suggest that influx and release pathways are major contributors to the transient component, whereas the lower sustained component is largely limited to influx pathways. The influx pathway during the transient probably involves an L-type Ca2+ channel that is regulated by tyrosine kinase activity. The pathways that participate in the sustained response are different because they are insensitive to l-channel antagonists and only slightly inhibited by genistein. The transient evoked in Ca2+-free media was blocked by genistein, inhibited by caffeine, and prevented by thapsigargin. Ionomycin-induced release of Ca2+ was unaffected by genistein, reduced by caffeine, and essentially eliminated by thapsigargin. Therefore, thapsigargin-mediated suppression of serotonin-activated release probably reflects depletion of Ca2+ from the sarcoplasmic reticulum, whereas genistein-mediated suppression probably reflects inhibition of tyrosine kinase linked release. Caffeine-mediated suppression appears to involve both partial depletion of Ca2+ and interference with release. Each A10 cell expressed at least two different ryanodine receptors and two different receptors for inositol 1,4,5-trisphosphate.

Protein tyrosine phosphorylation in smooth muscle: a potential coupling mechanism between receptor activation and intracellular calcium.

This review addresses a rapidly growing body of evidence suggesting that enhanced protein tyrosine phosphorylation may be a previously unrecognized mechanism for coupling receptor activation of vascular smooth muscle cells to increases In the intracellular concentration of Ca2+ and contraction. The hypothesis proposes that activation of diverse types of receptors that are not tyrosine kinase promotes stimulation of a cytosolic tyrosine kinase. In turn, the activated kinase induces tyrosine phosphorylation of substrates that are linked to regulatory mechanisms for release of intracellular Ca2+ stored in the sarcoplasmic reticulum and to regulatory mechanisms for influx of extracellular Ca2+. Within this framework, we examine some relevant functional aspects of receptor and nonreceptor tyrosine kinases in different types of cells, the emerging relationships between tyrosine kinase activity and regulation of intracellular Ca2+. We review studies of nonreceptor tyrosine kinase activity in vascular smooth muscle cells suggesting that a physiologically relevant kinase may be the enzyme called pp60. Data that appear to link tyrosine phosphorylation to contraction of smooth muscle are examined, particularly with respect to results obtained with tyrosine kinase inhibitors and measures of changes in tyrosine phosphorylation. Next, we review studies with cultured vascular smooth muscle cells that point to potential coupling between receptor activation, enhanced tyrosine phosphorylation of substrates such as the GTPase activating protein for ras, and the gamma-1 isoform of phospholipase C, and mechanisms controlling Ca2+ influx and release. Emphasis is placed on examining the strengths and weaknesses of different experimental approaches. Lastly, a summary of the data is provided which calls attention to some major issues requiring resolution to permit acceptance or rejection of the underlying hypothesis, and we briefly address some of its possible pathophysiological implications.

Coupling between [arginine8]-vasopressin-activated increases in protein tyrosine phosphorylation and cellular calcium in A7r5 aortic smooth muscle cells.

Effects of genistein, a tyrosine kinase inhibitor, on increases in [Ca2+]i and protein tyrosine phosphorylation induced by 20 nM [arginine 8]vasopressin (AVP) were studied in A7r5 aortic smooth muscle cells. In fura-2-loaded cells, AVP induced a rapid (0.5-2 min) transient increase in [Ca2+]i that was followed by a smaller sustained increase in [Ca2+]i. In 66% of the cells, the transient response involved both influx of extracellular Ca2+ and release of intracellular Ca2+: influx accounted for 6% of the response, and release accounted for 40%. However, in 34% of the cells, the relative contribution of influx and release during the transient could not be assessed. In all cells, the smaller sustained response was entirely dependent on extracellular Ca2+. Genistein (148 microM) always blocked the transient and sustained components of the Ca2+ response showing that both influx and release were genistein-sensitive. Isobestic fluorescence analysis, in medium containing 0.5 mM Mn2+ in place of Ca2+, showed that the influx pathway was selective because it did not conduct Mn2+. It also confirmed that Ca2+ release was blocked by genistein. In contrast, 105 microM lavendustin A, a different tyrosine kinase inhibitor, suppressed the transient by only 30%. Another inhibitor, tyrphostin 47 (80 microM), did not alter the transient or sustained components of the Ca2+ response. No AVP-induced increases in tyrosine phosphorylation were detected unless special procedures were used. When cells were preincubated in 10 mM vanadate, a tyrosine phosphatase inhibitor, AVP induced a transient increase in tyrosine phosphorylation (5-60 s). The time course for AVP-induced phosphorylation was similar to that for increase in [Ca2+]i. Vanadate alone increased tyrosine phosphorylation and induced a slow small increase in [Ca2+]i that was dependent on extracellular Ca2+. Genistein blocked tyrosine phosphorylation induced by AVP and vanadate, and it blocked the increase in [Ca2+]i induced by vanadate alone. In contrast, lavendustin or tyrphostin unexpectedly enhanced tyrosine phosphorylation induced by vanadate alone and precluded assessment of AVP-induced tyrosine phosphorylation in the presence of vanadate. Lavendustin produced time-dependent enhancement of vanadate-induced increase in [Ca2+]i. These results underscored the need for measuring cellular changes in protein tyrosine phosphorylation to assess potential functions of tyrosine kinase activity. Under conditions where changes in phosphorylation could be measured, the results suggested that AVP-activated increases in tyrosine phosphorylation may be coupled to AVP-activated mechanisms that regulate influx of extracellular Ca2+ and release of intracellular Ca2+.

Receptor-activated increases in intracellular calcium and protein tyrosine phosphorylation in vascular smooth muscle cells.

We studied the effects of protein tyrosine kinase inhibitors (genistein and tyrphostin) on receptor-activated increases in cellular Ca2+ ([Ca2+]i), and protein tyrosine phosphorylation in cultured canine femoral arterial smooth muscle cells. Fura-2 imaging analysis showed that each agonist evoked a transient increase in ([Ca2+]i) followed by a sustained plateau phase. Experiments in Ca(2+)-free medium showed that 70-80% of the transient increase in [Ca2+]i evoked by either agonist is due to influx of extracellular Ca2+ whereas the plateau phase is only due to Ca2+ entry. Pre-incubation with genistein or tyrphosin markedly inhibited the transient rise in [Ca2+]i evoked by serotonin or phenylephrine. Immunoblot analysis of cell extracts with antiphosphotyrosine antibodies revealed that serotonin and phenylephrine also evoked an increase in tyrosine phosphorylation of several substrates. These increases were abolished by tyrosine kinase inhibitors. One of the major substrates was recognized by an an antibody for rasGAP. These data suggest that receptor-activated increases in [Ca2+]i in vascular smooth muscle cells may be coupled to receptor-activated increases in protein tyrosine phosphorylation.

Modulation of Ca2+ sensitivity in smooth muscle by genistein and protein tyrosine phosphorylation.

Genistein, a potent tyrosine kinase inhibitor, inhibits contraction of several types of smooth muscle, suggesting that protein tyrosine phosphorylation may be an important regulatory mechanism for smooth muscle contraction. We suspected that one site between activation of smooth muscle and contraction which might be modulated by protein tyrosine phosphorylation involved mechanisms for control of Ca2+ sensitivity. Since smooth muscle permeabilized with staphylococcal alpha-toxin permits direct assessment of agonist-induced Ca2+ sensitivity, we studied the effects of genistein on potential coupling between tyrosine phosphorylation and Ca2+ sensitivity in permeabilized ileal smooth muscle. Results show that contraction of intact preparations with carbachol is markedly and reversibly inhibited by 40% at 4 micrograms genistein/ml and by 60% at 20 micrograms genistein/ml. Permeabilized preparations that are contracted with a submaximal [Ca2+] in the presence of GTP relax when genistein is added to the medium. Genistein also reversibly inhibits contractions induced in permeabilized muscle with either a submaximal or maximal [Ca2+] in the presence of GTP, as well as receptor-coupled activation of Ca2+ sensitization with 10 microM carbachol/10 microM GTP. Activation of permeabilized preparations at pCa 4.6 in the presence of 100 microM GTP promotes time-dependent tyrosine phosphorylation of several substrates. Both phosphorylation and force are inhibited by genistein. However, relatively high levels of myosin light chain phosphorylation persist during genistein-induced inhibition of Ca2+ sensitivity. In contrast, genistein has no effect on Ca(2+)-activated contraction in Triton-skinned preparations in either the presence or the absence of GTP. This shows that it does not directly inhibit actin-myosin interaction and suggests that its target(s) may be a cytosolic or membrane-bound regulatory protein(s) that is leached from the preparations during Triton-skinning. Taken together, these new data suggest that (a) tyrosine phosphorylation of one or more substrates may be coupled to mechanisms which regulate Ca2+ sensitivity and (b) the inhibitory effects of genistein are probably due to inhibition of agonist-induced Ca2+ sensitivity.

Protein tyrosine phosphorylation, cellular Ca2+, and Ca2+ sensitivity for contraction of smooth muscle.

Our studies are guided by the novel hypothesis that protein tyrosine phosphorylation is an important mechanism for regulating contraction of smooth muscle. Several lines of evidence are reviewed which suggest that enhanced tyrosine phosphorylation participates in mechanisms that regulate cytosolic Ca2+ and Ca2+ sensitivity for contraction. First, vanadate-induced contraction of guinea-pig taenia coli is functionally linked to enhanced protein tyrosine phosphorylation of at least three substrates, apparently resulting from vanadate-mediated inhibition of protein tyrosine phosphatase activity. Second, vanadate-induced contraction is dependent on extracellular Ca2+. Third, increases in cytosolic Ca2+ resulting from stimulation of alpha 1-adrenergic receptors in cultured canine vascular smooth muscle cells are associated with enhanced tyrosine phosphorylation and are inhibited by genistein, a potent inhibitor of tyrosine kinase activity. Fourth, genistein markedly and reversibly suppresses Ca2+ sensitivity for contraction in ileal longitudinal smooth muscle permeabilized with staphylococcal alpha-toxin. Moreover, the same or similar substrates (e.g., 42-45, 70, 80-85, 95, 100, 110, 116, and 205 kDa) are tyrosine phosphorylated in response to Ca2+ or stimulation of muscarinic or alpha 1-adrenergic receptors. Collectively, these data strongly suggest that tyrosine phosphorylation is an important mechanism for regulation of smooth muscle contraction.

Vanadate-induced contraction of smooth muscle and enhanced protein tyrosine phosphorylation.

This study was guided by the hypothesis that protein tyrosine phosphorylation may participate in signal transduction that is associated with contraction of smooth muscle. Accordingly, because sodium vanadate stimulates contraction of smooth muscle and inhibits protein tyrosine phosphatases, we sought to determine if vanadate-induced contraction was linked to enhanced protein tyrosine phosphorylation. Pronounced and time-dependent increases (5- to 20-fold) occurred in tyrosine phosphorylation during vanadate-induced contraction of guinea pig taenia coli. Three substrates of 86, 116, and 205 kDa were tyrosine phosphorylated as detected by immunoblotting with phosphotyrosine monoclonal antibody. Vanadate-induced contraction and enhanced protein tyrosine phosphorylation were both reduced in the presence of 2.5-5 micrograms/ml genistein, a potent tyrosine kinase inhibitor. In the continued presence of vanadate, chelation of extracellular Ca2+ with 5 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) caused relaxation. However, pronounced phosphorylation of the substrates persisted even though isometric force decreased to control level. In this setting, replacement of the muscle bathing medium with fresh vanadate and EGTA-free medium caused a prompt spontaneous contraction which was immediately followed by relaxation and dephosphorylation of the substrates. Similarly, blockade of Ca(2+)-entry sites with 10 mM LaCl3 also relaxed smooth muscle strips that had been contracted with 1.5 mM vanadate. However, unlike EGTA-induced relaxation, replacement of the medium with fresh vanadate and LaCl3-free medium did not cause a spontaneous contraction. Taken together, these results show that vanadate-induced contraction of smooth muscle is probably coupled to enhanced protein tyrosine phosphorylation. The results also suggest that tyrosine phosphorylation may participate in Ca(2+)-dependent signalling mechanisms which regulate contraction of smooth muscle.

Tyrosine kinase inhibitors suppress agonist-induced contraction in smooth muscle.

Because tyrosine kinases participate in diverse signalling pathways, we suspected that these enzymes might also participate in regulation of signal transduction in smooth muscle. Therefore, we studied the effects of geldanomycin, tyrphostin, and genistein, three structurally unrelated tyrosine kinase inhibitors, on receptor-mediated and depolarization-mediated contraction in three different types of smooth muscle. Contraction elicited by stimulation of muscarinic receptors with carbachol, or by stimulation of alpha-adrenergic receptors with norepinephrine or phenylephrine were markedly (> 80%) and reversibly inhibited by tyrosine-kinase inhibitors. In contrast, only slight inhibition (20%) occurred in contractions elicited by K(+)-induced depolorization. Moreover, tyrphostin did not inhibit direct Ca(2+)-mediated activation of the contractile apparatus in preparations permeabilized with beta-escin. These results suggest the novel hypothesis that tyrosine kinases participate in regulation of signal transduction that is associated with receptor-mediated contraction of smooth muscle.

The phospholamban phosphatase associated with cardiac sarcoplasmic reticulum is a type 1 enzyme.

Canine cardiac sarcoplasmic reticulum vesicles contain intrinsic protein phosphatase activity, which can dephosphorylate phospholamban and regulate calcium transport. This phosphatase has been suggested to be a mixture of both type 1 and type 2 enzymes (E. G. Kranias and J. Di Salvo, 1986, J. Biol. Chem. 261, 10,029-10,032). In the present study the sarcoplasmic reticulum phosphatase activity was solubilized with n-octyl-beta-D-glucopyranoside and purified by sequential chromatography on DEAE-Sephacel, polylysine-agarose, heparin-agarose, and DEAE-Sephadex. A single peak of phosphatase activity was eluted from each column and it was coincident for both phospholamban and phosphorylase a, used as substrates. The partially purified phosphatase could dephosphorylate the sites on phospholamban phosphorylated by either cAMP-dependent or calcium-calmodulin-dependent protein kinase(s). Enzymatic activity was inhibited by inhibitor-2 and by okadaic acid (I50 = 10-20 nM), using either phosphorylase a or phospholamban as substrates. The sensitivity of the phosphatase to inhibitor-2 or okadaic acid was similar for the two sites on phospholamban, phosphorylated by the cAMP-dependent and the calcium-calmodulin-dependent protein kinases. Phospholamban phosphatase activity was enhanced (40%) by Mg2+ or Mn2+ (3 mM) while Ca2+ (0.1-10 microM) had no effect. These characteristics suggest that the phosphatase associated with cardiac sarcoplasmic reticulum is a type 1 enzyme, and this activity may participate in the regulation of Ca2+ transport through dephosphorylation of phospholamban in cardiac muscle.

Human retinal vascular cells differ from umbilical cells in synthetic functions and their response to glucose.

Cell culture systems have commonly been used to study mechanisms implicated in the pathogenesis of diabetic retinopathy, but the great majority of cell preparations used have been either of nonhuman retinal origin or nonretinal human origin. Because of questions of species and organ specificity in the function of cells of vascular origin, in this study, cultured microvascular endothelial cells (HREC), pericytes (HRPC), and pigment epithelial cells from the postmortem human retina, and endothelial cells from human umbilical vein (HUVEC) were evaluated with respect to cell proliferation, and secretory products potentially important in diabetic retinopathy, i.e., prostaglandins (PG) and plasminogen activators (PA), normalized to DNA content/well, under both basal (5 mM) and high (25 mM) glucose conditions. Glucose (25 mM) reduced DNA content similarly in both types of endothelial cells, had a lesser effect on HRPC, and did not significantly alter the proliferation of pigment epithelial cells. Basal secretion of PGI2 (measured as 6-keto-PGF1 alpha) was in the order HRPC much greater than HREC greater than HUVEC, whereas PGE2 secretion was in the order HREC much greater than HRPC greater than HUVEC. Glucose (25 mM) stimulated PGI2 secretion by HRPC, but not by either type of endothelial cell, and enhanced PGE2 secretion by HREC, but not by HUVEC or HRPC. Release of plasminogen activator activity differed between HUVEC and HREC under basal conditions and addition of 25 mM glucose stimulated release only from HREC. Glucose (25 mM) stimulated PA secretion by HREC, but not by HUVEC. These findings provide evidence that human retinal pericytes are an important source of prostacyclin, and that there are differences between HREC and HUVEC with respect to secretory functions and their modulation by glucose, indicating regional specificity of these functions. Extrapolation to human retinal vascular cells from experiments using cells from heterologous vascular beds to draw inferences about the pathophysiology of diabetic retinopathy are not valid for these cellular functions.

Localization of acidic fibroblast growth factor within the mouse brain using biochemical and immunocytochemical techniques.

The localization of acidic fibroblast growth factor (aFGF) in the male mouse brain was studied with biochemical and immunocytochemical techniques. Using two peptide-based aFGF antisera directed against independent epitopes, Western gel analysis of dissected brain demonstrated significant levels of aFGF immunoreactivity in the pons-medulla, hypothalamus and cerebellum. The cortex contained much less immunoreactivity. Consistent with the biochemical data, immunocytochemical analysis with the same two antisera demonstrated that aFGF immunoreactivity is localized in neuronal cell bodies in these regions. Numerous immunoreactive neurons were observed in the reticular formation of the pons and medulla, as well as in several other brainstem nuclei and areas. Immunoreactive neurons were also present in the lateral and medial hypothalamus, and some thalamic, subthalamic and epithalamic nuclei. In the basal ganglia, immunoreactive neurons were present in the amygdala and septum. Few intensely stained immunoreactive neurons were observed in the striatum, pallidum and neocortex. Limbic cortices contained more numerous immunoreactive neurons than neocortex. These results support the concept that aFGF is present in the brain, where it is heterogeneously distributed in neuronal cell bodies in regions involved in sensory, extrapyramidal motor, limbic and autonomic functions. The results are consistent with various neurotrophic, mitogenic, and neuromodulatory functions associated with aFGF in the mammalian central nervous system.