Andrew M. Kahn

Uric Acid Causes Vascular Smooth Muscle Cell Proliferation by Entering Cells via a Functional Urate Transporter

Background: Soluble uric acid stimulates vascular smooth muscle cell (VSMC) proliferation by activating mitogen-activated protein kinases, and stimulating COX-2 and PDGF synthesis. The mechanism by which uric acid enters the VSMC is not known. We hypothesized that uric acid enters via transporters similar to that observed in the kidney. Methods: We studied the uptake of uric acid into rat VSMC under polarized and depolarized conditions and in the presence of organic anion transport (OAT) inhibitors (probenecid and benzbromarone) or p-aminohippurate (PAH). We also examined the ability of probenecid to inhibit uric acid-induced VSMC proliferation and monocyte chemoattractant protein-1 (MCP-1) synthesis. Results:14C-Urate uptake was shown in VSMC and was enhanced under depolarized conditions. 14C-Uric acid uptake was inhibited by probenecid and benzbromarone, as well as by unlabelled urate and PAH. Probenecid blocked VSMC proliferation and MCP-1 expression in response to uric acid. VSMC did not express rOAT1-3, rOAT-5 or URAT-1 mRNA by PCR, but did express the voltage-sensitive transporter (UAT) by both PCR and RNase protection assay. Conclusions: Urate enters VSMC by both voltage-sensitive and OAT pathways, and the uptake, cell proliferation and MCP-1 expression can be blocked by OAT inhibitors. The specific transporter(s) responsible for the urate uptake remains to be determined.

Insulin reduces contraction and intracellular calcium concentration in vascular smooth muscle.

Resistance to insulin-induced glucose disposal is associated with hypertension, in accord with recent reports that insulin-induced vasodilation is impaired in men with resistance to insulin-induced glucose disposal. Nevertheless, the mechanism of insulin-induced vasodilation is not known. We wished to determine whether a physiological concentration of insulin inhibits agonist-induced contraction at the level of the individual vascular smooth muscle cell, and if so, how. Dispersed vascular smooth muscle cells from dog femoral artery were grown on collagen gels for 4 to 8 days. Contraction and intracellular Ca2+ concentration of individual cells were measured by photomicroscopy and fura 2 epifluorescence microscopy, respectively. Serotonin and angiotensin II contracted cells in a dose-dependent manner. Preincubation of cells for 20 minutes (short-term) or 7 days (long-term) with insulin (40 microU/mL) inhibited serotonin- and angiotensin II-induced contractions by approximately 50%. Insulin (10 microU/mL) acutely inhibited serotonin-induced contraction by 34%. The maximal effect of high extracellular K(+)-induced contraction was not affected by short-term insulin exposure, but the ED50 for extracellular K(+)-induced contraction was increased from 7.6 +/- 2.5 to 16.0 +/- 3.9 mmol/L (P < .05). Short-term insulin exposure also attenuated the peak rise of the serotonin-induced intracellular Ca2+ transient and increased the rate constant for intracellular Ca2+ decline. Verapamil and ouabain completely blocked the attenuation of agonist-induced contraction by short-term insulin exposure, indicating the importance of voltage-operated Ca2+ channels and the Na(+)-K+ pump for this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevalence of Kawasaki Disease in Young Adults With Suspected Myocardial Ischemia

Background— Up to 25% of patients with untreated Kawasaki disease (KD) and 5% of those treated with intravenous immunoglobulin will develop coronary artery aneurysms. Persistent aneurysms may remain silent until later in life when myocardial ischemia can occur. We sought to determine the prevalence of coronary artery aneurysms suggesting a history of KD among young adults undergoing coronary angiography for evaluation of possible myocardial ischemia. Methods and Results— We reviewed the medical histories and coronary angiograms of all adults <40 years of age who underwent coronary angiography for evaluation of suspected myocardial ischemia at 4 San Diego hospitals from 2005 to 2009 (n=261). History of KD-compatible illness and cardiac risk factors were obtained by medical record review. Angiograms were independently reviewed for the presence, size, and location of aneurysms and coronary artery disease by 2 cardiologists blinded to the history. Patients were evaluated for number of risk factors, angiographic appearance of their coronary arteries, and known history of KD. Of the 261 young adults who underwent angiography, 16 had coronary aneurysms. After all clinical criteria were assessed, 5.0% had aneurysms definitely (n=4) or presumed (n=9) secondary to KD as the cause of their coronary disease. Conclusions— Coronary sequelae of KD are present in 5% of young adults evaluated by angiography for myocardial ischemia. Cardiologists should be aware of this special subset of patients who may benefit from medical and invasive management strategies that differ from the strategies used to treat atherosclerotic coronary artery disease.

Role of JAK/STAT pathway in IL-6-induced activation of vascular smooth muscle cells

Background/Aims: IL-6, an inducer of the acute-phase response, is linked with the development of vascular disease and atherosclerosis. One mechanism likely involves direct effects of IL-6 on vascular smooth muscle cells (VSMC), for IL-6 can induce VSMC proliferation and the release of monocyte chemoattractant protein-1 (MCP-1). We hypothesized that this stimulation occurs via the JAK (janus-activated kinase)/STAT (signal and transducers and activators of transcription) signaling pathway. Methods: Rat VSMC were stimulated with IL-6 in the presence or absence of a JAK 2 inhibitor, and the activation of STAT 3 (by Western), MCP-1 (by ELISA) and DNA synthesis (by 3H-thymidine incorporation) was determined. Results: IL-6 rapidly induced phosphorylation of STAT 3 in a dose- and time-dependent manner with a peak expression at 30 min. IL-6 also stimulated MCP-1 protein production and DNA synthesis dose dependently. 50 µM of AG490, a specific JAK 2 inhibitor, partially inhibited STAT 3 activation and MCP-1 production, with near complete inhibition of DNA synthesis. Conclusion: The JAK/STAT pathway partially mediates IL-6-induced MCP-1 production and DNA synthesis in rat VSMC. These studies implicate a role of the JAK/STAT pathway in the development of vascular disease and atherosclerosis.

Insulin Acutely Inhibits Cultured Vascular Smooth Muscle Cell Contraction by a Nitric Oxide Synthase–Dependent Pathway

Insulin acutely decreases contractile agonist-induced Ca2+ influx and contraction in endothelium-free cultured vascular smooth muscle (VSM) cells, but the mechanism is not known. Since it has been reported that insulin-induced vasodilation in humans is linked to nitric oxide synthase activity, we wished to determine whether insulin inhibits Ca2+ influx and contraction of cultured vascular smooth muscle cells by a nitric oxide synthase-dependent pathway. Primary cultures of endothelial cell-free VSM cells from canine femoral artery were preincubated with and without 1 nmol/L insulin for 30 minutes, and the 5-minute production of cGMP was measured. Insulin alone did not affect cGMP production, but in the presence of 10(-5) mol/L serotonin insulin stimulated cGMP production by 60%. N(G)-monomethyl-L-arginine (0.1 mmol/L), an inhibitor of nitric oxide synthase, inhibited the conversion of arginine to citrulline by these cells, blocked insulin-stimulated cGMP production, and blocked the inhibition by insulin of 5-hydroxytryptamine (5-HT)-stimulated Mn+2 (a Ca2+ surrogate) influx and contraction. Insulin did not affect contraction of VSM cells grown under conditions designed to deplete the cells of tetrahydrobiopterin, an essential cofactor of nitric oxide synthase. These studies demonstrate that insulin acutely inhibits 5-HT-stimulated Ca2+ influx and contraction of endothelium-free cultured VSM cells by a nitric oxide synthase-dependent mechanism.

Insulin-Stimulated Glucose Transport Inhibits Ca2+ Influx and Contraction in Vascular Smooth Muscle

BACKGROUNDnInsulin attenuates serotonin-induced Ca2+ influx, the intracellular Ca2+ transient, and contraction of cultured vascular smooth muscle cells from dog femoral artery. These studies were designed to test whether insulin-induced glucose transport was an early event leading to the inhibitory effects of insulin on Ca2+ influx, intracellular Ca2+ concentration, and contraction in these cells.nnnMETHODS AND RESULTSnInsulin 1 nmol/L stimulated the 30-minute uptake of [3H]2-deoxyglucose in these cells via a phloridzin-inhibitable mechanism. Contraction of individual cells was measured by photomicroscopy, intracellular Ca2+ concentration was monitored by measuring fura 2 fluorescence by use of Ca(2+)-sensitive excitation wavelengths, and Ca2+ influx was estimated by the rate of Mn2+ quenching of intracellular fura 2 fluorescence when excited at a Ca(2+)-insensitive wave-length. In the presence of 5 mmol/L glucose, preincubation of cells for 30 minutes with 1 nmol/L insulin inhibited 10(-5) mol/L serotonin-induced contraction of individual cells by 62% (P < .01) and decreased the serotonin-stimulated component of Mn2+ influx by 78% (P < .05). Removing glucose from the preincubation medium or adding 1 mmol/L phloridzin completely eliminated these effects of insulin. Insulin lowered the serotonin-induced intracellular Ca2+ peak by 37% (P < .05), and phloridzin blocked this effect of insulin. When glucose uptake was increased to the insulin-stimulated level by preincubation of the cells for 30 minutes with 25 mmol/L glucose in the absence of insulin, serotonin failed to stimulate Mn2+ influx, the serotonin-induced Ca2+ peak was decreased by 46% (P < .05), serotonin-induced contraction was inhibited by 60% (P < .01), and addition of insulin did not further inhibit contraction.nnnCONCLUSIONSnSince the effects of insulin on serotonin-stimulated Ca2+ transport, intracellular Ca2+ concentration, and contraction were dependent on glucose transport and were duplicated when glucose transport was stimulated by high extracellular glucose concentration rather than insulin per se, it is concluded that insulin-stimulated glucose transport is an early event that leads to decreased Ca2+ influx and contraction in vascular smooth muscle.

Insulin-stimulated cyclic guanosine monophosphate inhibits vascular smooth muscle cell migration by inhibiting Ca/calmodulin-dependent protein kinase II.

Background—Insulin resistance is associated with vascular disease. Physiological concentrations of insulin inhibit cultured vascular smooth muscle cell (VSMC) migration in the presence of nitric oxide, and the failure to do so in insulin-resistant states may aggravate vascular disease. We sought to determine the molecular mechanisms by which insulin inhibits VSMC migration. Methods and Results—Insulin at 1 nmol/L stimulated cGMP production in cultured rat VSMCs that were induced to express inducible nitric oxide synthase (iNOS). VSMC migration was measured in a wound-closure assay, and the platelet-derived growth factor-AB (PDGF-AB)-stimulated component of VSMC migration after wounding was inhibited by insulin, 8-Br-cGMP, and 1-[N-0-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62), a selective inhibitor of calcium/calmodulin-dependent protein kinase II (CaM kinase II). Wounding alone or incubating cells with only PDGF-AB stimulated CaM kinase II activity in an insulin- and 8-Br-cGMP-inhibitable manner. Transfecting VSMCs with a constitutively active CaM kinase II mutant blocked the inhibition by insulin of both wound-induced and wound plus PDGF-AB-induced VSMC migration. High intracellular Ca2+ ([Ca]i)-stimulated CaM kinase II activity was inhibited by 8-Br-cGMP by an okadaic acid-sensitive mechanism. Conclusions—We conclude that in cultured rat VSMCs expressing iNOS, insulin, via stimulation of cGMP production, inhibits both wound alone-induced and the PDGF-AB-stimulated component of VSMC migration by inhibiting CaM kinase II activity. cGMP inhibits CaM kinase II at a post-[Ca]i step by a protein phosphatase-dependent mechanism.

Insulin inhibits migration of vascular smooth muscle cells with inducible nitric oxide synthase.

Vascular smooth muscle cell (VSMC) migration participates in atherosclerosis and arterial restenosis after balloon angioplasty. Because these processes are enhanced in insulin-resistant states, our goal was to determine whether insulin affects VSMC migration and, if so, how. The migration of primary cultured VSMCs from canine femoral artery was measured with the use of a wound migration assay and related to cGMP levels. Insulin (1 nmol/L) did not affect migration or cGMP production in control cells. When inducible nitric oxide synthase (iNOS) was induced by 24-hour preincubation with lipopolysaccharide and interleuken-1beta, basal migration decreased, cGMP production increased, and insulin inhibited migration by >90% and stimulated cGMP production by 3-fold. The nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine blocked the affect of insulin on the migration of VSMCs with iNOS. 8-Bromo-cGMP inhibited VSMC migration in control cells, and 1-H-1[1,2,4]oxadiazolo-[4, 3a]quinoxolin-1-one, a selective inhibitor of guanylate cyclase, blocked the inhibition by insulin of migration of cells with iNOS. We conclude that insulin does not normally affect cGMP production or the migration of these VSMCs. However, after the induction of iNOS, insulin stimulates cGMP production and inhibits migration via an NOS-and a cGMP-dependent mechanism.

Insulin inhibits serotonin-induced Ca2+ influx in vascular smooth muscle.

Insulin in physiological concentrations attenuates the agonist-induced intracellular Ca2+ ([Ca2+]i) transient and inhibits contraction in individual nonproliferated cultured canine femoral artery vascular smooth muscle cells (VSMCs). In the present study, we wished to define the effects of insulin on individual components of Ca2+ transport in vascular smooth muscle. Methods and ResultsInsulin (40 μU/mL) attenuated the 5-hydroxytryptamine (5-HT, serotonin; 10−5 mol/L)-induced [Ca2+]i transient (measured by fura 2 fluorescence) in primary confluent canine femoral artery VSMCs in the presence of extracellular Ca2+. In Ca2+ -free media, the 5-HT-induced [Ca2+ transient was reduced by 42% and was not affected by insulin. This finding suggested that insulin inhibits 5-HTinduced Ca2+ influx but does not affect sarcolemmal Call efflux or Ca2+ release from intracellular stores. In support of those conclusions, we found that insulin inhibited the 5-HT-induced component of Mn2+ (a Ca2+ surrogate) influx (measured by fura 2 fluorescence quenching at the Ca2+ isosbestic excitation wavelength). In addition, 5-HT stimulated the rates of 45Ca2+ efflux from intact cells (a measure of sarcolemmal Ca2+ efflux) and from saponin-permeabilized cells (a measure of Ca2+ release from intracellular stores), but insulin did not affect these rates of 45Ca2+ efflux. ConclusionsWe conclude that a physiological insulin concentration attenuates the 5-HT- induced [Ca2+]i transient in confluent primary cultured canine femoral artery VSMCs by inhibiting the 5 -HT-induced component of Ca2+ influx but not by affecting sarcolemmal Ca2+ efflux or Ca2+ release from intracellular stores.

Difference between human red blood cell Na+-Li+ countertransport and renal Na+-H+ exchange.

Several laboratories have reported that the activities of sodium-lithium countertransport are increased in red blood cells from patients with essential hypertension. Based on the many similarities between this transport system and the renal sodium-proton exchanger, a hypothesis has been put forth in the literature that increased red blood cell sodium-lithium countertransport activity may be a marker for increased sodium-proton exchange activity in the renal proximal tubule. The present studies were designed to test the hypothesis that sodium-lithium countertransport in red blood cells from humans or rabbits is mediated by the same transport mechanism that mediates sodium-proton exchange in the renal brush border from those species. Similar to what has been reported for the rabbit, the present studies show that an amiloride-sensitive sodium-proton exchanger is present in human renal brush border vesicles. However, Na+-Li+ countertransport in human and rabbit red blood cells, assayed under several different conditions, was not inhibited by amiloride. In agreement with what has been reported for humans, the present studies show that extracellular proton-stimulated sodium efflux is inhibited by amiloride in rabbit red blood cells. These data demonstrate a difference (amiloride sensitivity) between the red blood cell sodium-lithium countertransporter and the renal brush border sodium-proton exchanger in humans and rabbits. These experiments detract from the hypothesis that increased red blood cell sodium-lithium countertransport activity in patients with essential hypertension is a marker for increased sodium-proton exchange activity in the renal brush border.