The novel endocannabinoid receptor GPR55 is activated by atypical cannabinoids but does not mediate their vasodilator effects.

BACKGROUND AND PURPOSE: Atypical cannabinoids are thought to cause vasodilatation through an as-yet unidentified 'CBx' receptor. Recent reports suggest GPR55 is an atypical cannabinoid receptor, making it a candidate for the vasodilator 'CBx' receptor. The purpose of the present study was to test the hypothesis that human recombinant GPR55 is activated by atypical cannabinoids and mediates vasodilator responses to these agents. EXPERIMENTAL APPROACH: Human recombinant GPR55 was expressed in HEK293T cells and specific GTPgammaS activity was monitored as an index of receptor activation. In GPR55-deficient and wild-type littermate control mice, in vivo blood pressure measurement and isolated resistance artery myography were used to determine GPR55 dependence of atypical cannabinoid-induced haemodynamic and vasodilator responses. KEY RESULTS: Atypical cannabinoids O-1602 and abnormal cannabidiol both stimulated GPR55-dependent GTPgammaS activity (EC50 approximately 2 nM), whereas the CB1 and CB2-selective agonist WIN 55,212-2 showed no effect in GPR55-expressing HEK293T cell membranes. Baseline mean arterial pressure and heart rate were not different between WT and GPR55 KO mice. The blood pressure-lowering response to abnormal cannabidiol was not different between WT and KO mice (WT 20+/-2%, KO 26+/-5% change from baseline), nor was the vasodilator response to abnormal cannabidiol in isolated mesenteric arteries (IC50 approximately 3 micro M for WT and KO). The abnormal cannabidiol vasodilator response was antagonized equivalently by O-1918 in both strains. CONCLUSIONS: These results demonstrate that while GPR55 is activated by atypical cannabinoids, it does not appear to mediate the vasodilator effects of these agents.

P2X1 stimulation promotes thrombin receptor-mediated platelet aggregation.

P2X1 receptors are ATP-gated channel demonstrated to be involved in multiple platelet responses, although in vitro analysis has been complicated by the effects of rapid desensitization. To further investigate potential roles of P2X1 receptors in platelet activation, the current study employed methods which maximally preserved P2X1 functionality. In preliminary in vivo studies, P2X1-deficiency reduced thrombus formation following the laser-induced, but not FeCl3-induced injury. Given the multiple potential mechanisms involved in thrombus formation in vivo, including tissue-factor/thrombin generation pathways, subsequent studies were designed to investigate the effects of P2X1 inhibition or stimulation on platelet activation in vitro; specifically, the interaction of P2X1 with thrombin receptor stimulation. Aggregation initiated by low/threshold levels of a protease-activated receptor (PAR)4 agonist was reduced in P2X1-deficient murine platelets, and inhibition of P2X1 in wild-type platelets similarly reduced PAR4-mediated aggregation. In human platelets, aggregation to low/threshold stimulation of PAR1 was inhibited with the P2X1 antagonist MRS2159. In addition, P2X1 stimulation primed human platelet responses, such that subsequent sub-threshold PAR1 responses were converted into significant aggregation. Selective ADP receptor inhibitors attenuated P2X1-mediated priming, suggesting that the synergy between P2X1 and sub-threshold PAR1 stimulation was in part because of enhanced granular release of ADP. Overall, the present study defines a novel interaction between platelet P2X1 and thrombin receptors, with P2X1 functioning to amplify aggregation responses at low levels of thrombin receptor stimulation.

Urotensin II and renal function in the rat.

Urotensin II (UII) is a potent vasoactive hormone in mammals. However, despite its well-known effects on epithelial sodium transport in fish, little is known about its actions on the mammalian kidney. The aim of this study was to determine the effects of UII on renal function in the rat. Using standard clearance methods, the effects of rUII and the rat UII receptor (UT) antagonist, urantide, were studied. UII was measured in plasma and urine by radioimmunoassay. UII and UT were localized in the kidney by immunohistochemistry and mRNA expression quantified. Rat urinary [UII] was 1,650-fold higher than that in plasma. Immunoreactive-UII was localized to the proximal tubules, outer and inner medullary collecting ducts (IMCD); UT receptor was identified in glomerular arterioles, thin ascending limbs, and IMCD. UII and UT mRNA expression was greater in the medulla; expression was higher still in spontaneously hypertensive rats (SHRs) associated with raised plasma (UII). Injection of rUII induced reductions in glomerular filtration rate (GFR), urine flow, and sodium excretion. Urantide infusion resulted in increases in these variables. Endogenous UII appears to contribute to the regulation of GFR and renal sodium and water handling in the rat. While hemodynamic changes predominate, we cannot rule out the possibility of a direct tubular action of UII. Increased expression of UII and UT in the SHR suggests that UII plays a role in the pathophysiology of cardiovascular disease.

Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide.

Angiotensin II (ANG II) is a pleiotropic vasoactive peptide that binds to two distinct receptors: the ANG II type 1 (AT(1)) and type 2 (AT(2)) receptors. Activation of the renin-angiotensin system (RAS) results in vascular hypertrophy, vasoconstriction, salt and water retention, and hypertension. These effects are mediated predominantly by AT(1) receptors. Paradoxically, other ANG II-mediated effects, including cell death, vasodilation, and natriuresis, are mediated by AT(2) receptor activation. Our understanding of ANG II signaling mechanisms remains incomplete. AT(1) receptor activation triggers a variety of intracellular systems, including tyrosine kinase-induced protein phosphorylation, production of arachidonic acid metabolites, alteration of reactive oxidant species activities, and fluxes in intracellular Ca(2+) concentrations. AT(2) receptor activation leads to stimulation of bradykinin, nitric oxide production, and prostaglandin metabolism, which are, in large part, opposite to the effects of the AT(1) receptor. The signaling pathways of ANG II receptor activation are a focus of intense investigative effort. We critically appraise the literature on the signaling mechanisms whereby AT(1) and AT(2) receptors elicit their respective actions. We also consider the recently reported interaction between ANG II and ceramide, a lipid second messenger that mediates cytokine receptor activation. Finally, we discuss the potential physiological cross talk that may be operative between the angiotensin receptor subtypes in relation to health and cardiovascular disease. This may be clinically relevant, inasmuch as inhibitors of the RAS are increasingly used in treatment of hypertension and coronary heart disease, where activation of the RAS is recognized.

Biosynthetic ganciclovir triphosphate: its isolation and characterization from ganciclovir-treated herpes simplex thymidine kinase-transduced murine cells.

A method is described for the preparation of ganciclovir triphosphate (GCV-TP) using murine colon cancer cells (MC38) transduced with the herpes simplex virus-thymidine kinase (MC38/HSV-tk). Murine cells transduced with viral-tk contain required viral and host enzymes needed for complete cellular synthesis of this potent antiviral metabolite. Dose response studies showed optimal intracellular levels of GCV-TP occurred after exposure of MC38/HSV-tk cells to 300 microM ganciclovir for 24 h producing 7.5 nmol GCV-TP/10(6) cells. This reflects cellular accumulation of GCV-TP to levels 25-fold greater than the medium concentration of parent drug. A simple isolation scheme included methanolic extraction and anion-exchange chromatography to recover the target triphosphate. Mass spectral analysis and selective enzyme degradation provided structural confirmation of the purified product. Biological activity of the purified GCV-TP was demonstrated by competitive inhibition experiments using human DNA polymerase alpha and HSV DNA polymerase that showed substantially greater sensitivity for the viral polymerase in agreement with previous reports. The GCV-TP obtained was further used to enzymatically prepare GCV mono- and diphosphate in high yield. This method provides an easily scalable means of preparing milligram amounts of the triphosphates of pharmacologically active acyclic nucleosides like ganciclovir.

Spinal interneurons play a minor role in generating ongoing renal sympathetic nerve activity in spinally intact rats.

The purpose of the present study was to determine whether spinal interneurons play a role in the regulation of sympathetic activity in spinally intact rats. In acutely spinally transected rats, we have described a population of spinal interneurons that, by virtue of correlations between their ongoing firing rates and the magnitude of ongoing renal sympathetic nerve activity (RSNA), are candidates for generators of sympathetic activity. Further evidence for a sympathetic role for these neurons comes from our observation that cervical spinal stimulation that reduces RSNA also reduces their discharge rates. In chloralose-anesthetized, spinally intact and spinally transected rats, we recorded ongoing RSNA and the ongoing activities of T(10) dorsal horn and intermediate zone interneurons, and we determined the incidence of sympathetically related neurons in these rats by cross-correlating their activities with RSNA. The incidence of correlated neurons was much smaller in spinally intact than in spinally transected rats. We stimulated the dorsolateral, C(2-3) spinal cord before and after acute C(1) spinal transection. Dorsolateral cervical stimulation in spinally transected rats reduced both RSNA and the activities of most T(10) interneurons, but stimulation in spinally intact rats increased RSNA while still reducing the activities of most T(10) interneurons. Both the low incidence of sympathetically correlated spinal neurons in intact rats and the dissociation between the effects of cervical stimulation on RSNA and the discharge rates of spinal interneurons argue against these neurons playing a major role in regulating sympathetic activity in intact rats.

Role of NADPH oxidase in the vascular hypertrophic and oxidative stress response to angiotensin II in mice.

Oxygen-derived free radicals are involved in the vascular response to angiotensin II (Ang II), but the role of NADPH oxidase, its subunit proteins, and their vascular localization remain controversial. Our purpose was to address the role of NADPH oxidase in the blood pressure (BP), aortic hypertrophic, and oxidant responses to Ang II by taking advantage of knockout (KO) mice that are genetically deficient in gp91(phox), an NADPH oxidase subunit protein. The baseline BP was significantly lower in KO mice than in wild-type (WT) (92+/-2 [KO] versus 101+/-1 [WT] mm Hg, P<0.01), but infusion of Ang II for 6 days caused similar increases in BP in the 2 strains (33+/-4 [KO] versus 38+/-2 [WT] mm Hg, P>0.4). Ang II increased aortic superoxide anion production 2-fold in the aorta of WT mice but did not do so in KO mice. Aortic medial area increased in WT (0.12+/-0.02 to 0.17+/-0.02 mm(2), P<0.05), but did not do so in KO mice (0.10+/-0.01 to 0.11+/-0.01 mm(2), P>0.05). Histochemistry and polymerase chain reaction demonstrated gp91(phox) localized in endothelium and adventitia of WT mice. Levels of reactive oxidant species as indicated by 3-nitrotyrosine immunoreactivity increased in these regions in WT but not in KO mouse aorta in response to Ang II. These results indicate an essential role in vivo of gp91(phox) and NADPH oxidase-derived superoxide anion in the regulation of basal BP and a pressure-independent vascular hypertrophic and oxidant stress response to Ang II.

Glucocorticoids inhibit tetrahydrobiopterin-dependent endothelial function.

Tetrahydrobiopterin (BH4) acts as an important co-factor for endothelial nitric oxide synthase (eNOS). Glucocorticoids have been shown to inhibit expression of the rate-limiting enzyme for tetrahydrobiopterin synthesis, GTP cyclohydrolase, in other cell types. We hypothesized that endothelium-dependent vasodilator responses would be blunted in rats made hypertensive with dexamethasone. Further, we hypothesized that treatment of rat vascular segments with dexamethasone would result in attenuation of endothelial function accompanied by decreased GTP cyclohydrolase expression. We report that endothelium-dependent relaxation responses to the calcium ionophore A23187 are reduced in aortic rings from dexamethasone-hypertensive rats compared with sham values. Dexamethasone incubation abolishes contraction to Nomega-nitro-L-arginine (L-NNA, 10(-5) M) in endothelium-intact aortic rings, and inhibits expression of GTP cyclohydrolase. We conclude that inhibition of BH4 synthesis by glucocorticoid regulation of GTP cyclohydrolase expression may contribute to reduced endothelium-dependent vasodilation characteristic of glucocorticoid-induced hypertension.

Impaired ceramide signalling in spontaneously hypertensive rat vascular smooth muscle: a possible mechanism for augmented cell proliferation.

OBJECTIVES: In hypertension, the vascular wall undergoes morphological changes that alter mechanical responses to vasoactive substances. Ceramide is a recently identified second messenger synthesized in response to cytokines such as tumour necrosis factor alpha (TNF-alpha). It has been previously demonstrated that vascular smooth muscle cells (VSMC) from genetically hypertensive rats proliferate at a higher rate than those of normotensive origin. We tested the hypothesis that the ceramide pathway is impaired in VSMC from spontaneously hypertensive rats (SHR). DESIGN: VSMC were isolated from aortae of SHR and from Wistar-Kyoto (WKY) rats. Ceramide levels were measured under baseline and agonist-stimulated conditions and cell proliferation was monitored. METHODS: Cell proliferation was determined by cell counting. Ceramide levels were determined via radioactive labelling, high-performance thin-layer chromatography and phosphorimaging. Relative mRNA levels of neutral sphingomyelinase were determined using semi-quantitative polymerase chain reaction (PCR). RESULTS: Basal ceramide levels in untreated cells were lower in cells from SHR compared to WKY rats. During chronic treatment with TNF-alpha, ceramide levels increased in WKY rat cells but remained unchanged in cells from SHR. TNF-alpha treatment had an inhibitory effect on WKY rat VSMC proliferation, but stimulated proliferation in cells from SHR. Short-term incubation with TNF-alpha resulted in a greater increase in ceramide in cells from WKY rats than those from SHR. Semiquantitative PCR analysis indicated that neutral sphingomyelinase mRNA may be reduced in SHR VSMC. CONCLUSIONS: We conclude that ceramide synthesis is impaired in vascular smooth muscle from SHR and may contribute to increased VSMC proliferation in hypertension.

Novel signaling pathways contributing to vascular changes in hypertension.

In hypertension, increased peripheral resistance maintains elevated levels of arterial blood pressure. The increase in peripheral resistance results, in part, from abnormal constrictor and dilator responses and vascular remodeling. In this review, we consider four cellular signaling pathways as possible explanations for these abnormal vascular responses: (1) augmented signaling via the epidermal growth factor receptor to cause remodeling of the cerebrovasculature; (2) reduced sphingolipid signaling leading to blunted vasodilation and increased smooth muscle proliferation; (3) increased signaling via Rho/Rho kinase leading to enhanced vasoconstriction, and (4) a relative state of microtubular depolymerization favoring vasoconstriction in hypertension. These novel cell signaling pathways provide new pharmacological targets to reduce total peripheral vascular resistance in hypertension.

Use of a herpes thymidine kinase/neomycin phosphotransferase chimeric gene for metabolic suicide gene transfer.

Metabolic suicide gene transfer is widely applied for gene therapy of cancer, and retroviral vectors expressing the herpes simplex virus thymidine kinase (HSV-tk) gene are commonly used in clinical trials. Most of these vectors contain positive selectable markers that undoubtedly facilitate the determination of viral titer and the identification of high-titer producer clones. However, the presence of additional transcriptional units may result in reduced expression of the gene of interest. The use of fusion genes expressing bifunctional proteins may help to overcome this problem. We have constructed a retroviral vector carrying the TNFUS69 chimeric gene, which originates from the fusion of the HSV-tk and neomycin phosphotransferase II genes, and evaluated the functional expression of the encoded fusion protein. In vitro, expression of the fusion gene conferred to target cells both resistance to neomycin and selective sensitivity to the antiherpetic drugs ganciclovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine. Cells transduced with the fusion gene, however, showed reduced ability to phosphorylate ganciclovir compared with cells expressing the native HSV-tk. Therefore, although the fusion gene may be used as a constituent of retroviral cassettes for positive and negative selection in vitro, its usefulness for suicide gene transfer applications in vivo may depend upon the possibility of using (E)-5-(2-bromovinyl)-2'-deoxyuridine in a clinical context.

Ongoing and stimulus-evoked activity of sympathetically correlated neurons in the intermediate zone and dorsal horn of acutely spinalized rats.

We have shown previously that in the acutely spinalized anesthetized rat the activities of many dorsal horn interneurons (DHN) at the T(10) level are correlated positively with both ongoing and stimulus-evoked renal sympathetic nerve activity (RSNA) and therefore may belong to networks generating RSNA after acute, cervical, spinal transection. In the present study, we recorded from both DHN and interneurons in the intermediate zone (IZN) of the T(10) spinal segment in acutely C(1)-transected, chloralose-anesthetized, artificially respired rats. The activities of a similar percentage of IZN and DHN were correlated positively with ongoing RSNA, but the peaks of spike-triggered averages of RSNA based on the activity of IZN were larger, relative to dummy averages, than spike-triggered averages of RSNA based on the activity of DHN. Sympathetically correlated DHN and IZN differed in their responses to noxious somatic stimuli. Most correlated DHN had relatively simple somatic fields; they were excited by noxious stimulation of the T(10) and nearby dermatomes and inhibited by stimulation of more distal dermatomes. As we have shown previously, the excitatory and inhibitory fields of these neurons were very similar to fields that, respectively, excited and inhibited RSNA. On the other hand, the somatic fields of 50% of sympathetically correlated IZN were significantly more complex, indicating a difference between either the inputs or the processing properties of IZN and DHN. Sympathetically correlated IZN and DHN also differed in their responses to colorectal distension (CRD), a noxious visceral stimulus. CRD increased RSNA in 11/15 rats and increased the activity of most sympathetically correlated T(10) IZN. On the other hand, CRD decreased the activity of a majority of sympathetically correlated T(10) DHN. These observations suggest that the same stimulus may differentially affect separate, putative, sympathoexcitatory pathways, exciting one and inhibiting the other. Thus the magnitude and even the polarity of responses to a given stimulus may be determined by the modality and location of the stimulus, the degree to which multiple pathways are affected by the stimulus, and the ongoing activity of presympathetic neurons, at multiple rostrocaudal levels, before stimulation. A multipathway system may explain the variability in autonomic responses to visceral and somatic stimuli exhibited in spinally injured patients.

Potentiation of the anti-HIV activity of zalcitabine and lamivudine by a CTP synthase inhibitor, 3-deazauridine.

Low levels of the CTP synthase inhibitor 3-deazauridine (3-DU) strongly potentiated the anti-HIV-1 activity of the 5'-triphosphates of the cytidine-based analogues [-]2'-deoxy-3'-thiacytidine (3TC; lamivudine) and 2',3'-dideoxycytidine (ddC). The potentiation was associated with a 3-DU-induced decrease in dCTP pool size; no changes were seen in cellular pool sizes of dATP, dGTP or dTTP.

Ceramide-induced vasorelaxation: An inhibitory action on protein kinase C.

Experiments were designed to examine the role of sphingosine, PP2A phosphatases, and protein kinase C (PKC) inhibition in mediating the vasodilatory effects of ceramide in rat thoracic aorta. Sphingosine did not cause vasorelaxation, and oleoylethanol-amine, a ceramidase inhibitor, did not affect sphingomyelinase-induced relaxation. Okadaic acid potentiated the relaxation response to ceramide. These observations rule out involvement of sphingosine and PP2A phosphatases in mediating ceramide-induced relaxation. Sphingomyelinase attenuated contractile and single-cell intracellular calcium responses to phorbol ester. Chelerythrine incubation potentiated the relaxation response to ceramide. These observations support a role for PKC inhibition in mediating the vasodilatory effects of ceramide.

Suppression of replication of multidrug-resistant HIV type 1 variants by combinations of thymidylate synthase inhibitors with zidovudine or stavudine.

The replication of recombinant multidrug-resistant HIV-1 clones modeled on clinically derived resistant HIV-1 strains from patients receiving long-term combination therapy with zidovudine (AZT) plus 2',3'-dideoxycytidine was found to regain sensitivity to AZT and stavudine (D4T) as a consequence of a pharmacologically induced decrease in de novo dTMP synthesis. The host-cell system used was phytohemagglutinin-stimulated peripheral blood mononuclear cells; dTMP and dTTP depletion were induced by single exposures to a low level of the thymidylate synthase inhibitor 5-fluorouracil (5-FU) or its deoxynucleoside, 2'-deoxy-5-fluorouridine. The host-cell response to the latter was biphasic: a very rapid decrease in the rate of de novo dTMP formation and, consequently, in intracellular dTTP pools, followed by slower recovery in both indices over 3 to 24 h. With the additional presence of AZT or D4T, however, replication of the multidrug-resistant HIV-1 strains remained inhibited, indicating dependence of HIV DNA chain termination by AZT-5'-monophosphate or 2',3'-didehydro-2', 3'-dideoxythymidine-5'-monophosphate in these resistant strains on simultaneous inhibition of host-cell de novo synthesis of thymidine nucleotides. No effect on viability of control (uninfected) phytohemagglutinin-stimulated/peripheral blood mononuclear cells was noted on 6-day exposures to 5-FU or 2'-deoxy-5-fluorouridine alone or in combination with AZT or D4T, even at drug levels severalfold higher than those used in the viral inhibition studies. These studies may provide useful information for the potential clinical use of AZT/5-FU or D4T/5-FU combinations for the prevention or reversal of multidrug resistance associated with long-term dideoxynucleoside combination therapy.

TNF-alpha-induced endothelium-independent vasodilation: a role for phospholipase A2-dependent ceramide signaling.

Ceramide is a novel second messenger generated by hydrolysis of membrane sphingomyelin by a neutral sphingomyelinase (nSMase). Cytokines such as tumor necrosis factor-alpha (TNF-alpha) have been shown to increase intracellular ceramide through phospholipase A2 (PLA2)-dependent activation of nSMase. TNF-alpha has been shown to cause endothelium-independent relaxation in isolated blood vessels. We have previously shown that exogenously applied sphingomyelinase and ceramide cause endothelium-independent vasodilation in rat thoracic aortas (D. G. Johns, H. Osborn, and R. C. Webb. Biochem. Biophys. Res. Commun. 237: 95-97, 1997). In the present study, we tested the hypothesis that ceramide mediates TNF-alpha-induced vasodilation. In phenylephrine-contracted rat thoracic aortic rings (no endothelium), TNF-alpha caused concentration-dependent relaxation in the presence of cyclooxygenase and lipoxygenase inhibitors. The phospholipase A2 antagonist 7,7-dimethyl-(5Z, 8Z)-eicosadienoic acid (DEDA; 50 microM) and the nonselective PLA2 antagonist quinacrine (30 microM) inhibited TNF-alpha-induced relaxation. In cultured rat aortic vascular smooth muscle cells, TNF-alpha (10(-7) g/ml) increased intracellular ceramide 1.5-fold over basal level (0.08 nmol/mg protein), which was blocked by the PLA2 antagonist DEDA (50 microM). We conclude that PLA2 activation and increased ceramide generation play a role in mediating TNF-alpha-induced endothelium-independent vasodilation.

Role of the M2 subunit of ribonucleotide reductase in regulation by hydroxyurea of the activity of the anti-HIV-1 agent 2',3'-dideoxyinosine.

The ribonucleotide reductase inhibitor hydroxyurea exhibits potent synergism, even at low, non-cytotoxic concentrations, with the anti-HIV-1 dideoxynucleoside 2',3'-dideoxyinosine, bringing about failure of HIV DNA synthesis and, thus, of HIV replication. To elucidate the incompletely defined role of hydroxyurea in the hydroxyurea/dideoxyinosine interaction and, in particular, to identify the reasons for the unusual selective inhibitory action of the combination on retroviral rather than on cellular DNA synthesis, we prepared specific cDNA probes to determine the effects of low-level hydroxyurea on mammalian cell ribonucleotide reductase M1 and M2 subunit mRNA, while simultaneously quantitating the effects of the drug on cell cycle and on deoxynucleoside triphosphate pools. While dTTP, dCTP, and dGTP pools changed little or even increased in the presence of low-level hydroxyurea, there took place a rapid and specific inhibition of M2-subunit-catalyzed generation of dATP, with consequent slowing of cellular DNA synthesis and prolongation of S phase. However, the latter effect, in turn, resulted in increased M2 subunit mRNA transcription (a process blocked in Go/G1-phase cells, with full-length functional M2 transcripts being generated only during S phase) and, hence, in a return to normal levels of dATP and to a normal rate of cellular DNA synthesis. Because of this self-regulating mechanism, hydroxyurea-induced host-cell toxicity was obviated under conditions where HIV DNA synthesis, a process sensitive to both dATP depletion and the chain-terminating properties of the other inhibitory component of the combination (ddATP derived from dideoxyinosine), was unable to recover.

The role of the endothelium in ceramide-induced vasodilation.

Ceramide, a novel sphingomyelin-derived second messenger mediates cellular signals of cytokines such as tumor necrosis factor-alpha (TNF-alpha). In the present study, we hypothesized that the endothelium contributes to ceramide-induced vasodilation. We report that relaxation to ceramide in endothelium-intact rat thoracic aortic rings is greater than in endothelium-denuded or endothelial nitric oxide synthase (endothelial NO synthase)-inactivated rings. We conclude that the endothelium contributes to ceramide-induced relaxation possibly through an interaction between sphingomyelin hydrolysis and endothelial NO synthase within caveolae.

Selective depletion of DNA precursors: an evolving strategy for potentiation of dideoxynucleoside activity against human immunodeficiency virus.

Human immunodeficiency virus type 1 (HIV-1) is wholly dependent on its host cell for a variety of essential metabolites. Among the latter are the deoxynucleoside-5'-triphosphates (dNTPs) required for reverse transcription of the single-stranded RNA viral genome into double-stranded viral DNA. Since viral DNA synthesis has an absolute requirement for all four dNTPs, restriction of a single one of these is sufficient to inhibit HIV-1 replication. To date, this therapeutic strategy has been most successful when depletion of the individual dNTP is coupled with exposure to its corresponding chain-terminating dideoxynucleoside (ddN). While several examples of such combined therapy have been defined and studied in vitro, that which has been investigated most extensively at both the laboratory and the clinical level is ddATP exposure combined with dATP depletion [with dATP restriction being induced by the ribonucleotide reductase inhibitor hydroxyurea (HU) and ddATP generated from its prodrug 2',3'-dideoxyinosine (ddI)]. Several long-term clinical trials of the hydroxyurea/2',3'-dideoxyinosine combination have been completed, with plasma viral RNA being reduced to undetectable levels in a substantial fraction (one-third to one-half) of the patients treated. The major advantages of this and analogous combinations discussed in this review are their low cost relative to other current multiple drug protocols and their potential for retention of activity against drug-resistant HIV mutants.