Does metformin affect outcomes in COVID-19 patients with new or pre-existing diabetes mellitus? A systematic review and meta-analysis.

AIMS: The COVID-19 pandemic is a global public health emergency and patients with diabetes mellitus (DM) are disproportionately affected, exhibiting more severe outcomes. Recent studies have shown that metformin is associated with improved outcomes in patients with COVID-19 and DM and may be a potential candidate for drug repurposing. We aimed to investigate the effects of metformin on outcomes in patients with COVID-19 and DM. METHODS: Databases (PubMed, Scopus, Web of Science, EMBASE, Clinicaltrials.gov and Cochrane library) were searched up to 10 April 2021 for studies reporting data on metformin use in COVID-19 patients with DM. The risk of bias was assessed using the Newcastle-Ottawa scale. Certainty of evidence was rated using the GRADE approach. The primary outcome was mortality reported as odds ratio (OR). A random-effects meta-analysis was carried out on both unadjusted and adjusted ORs. This study is registered with PROSPERO, CRD42020221842. RESULTS: In total, 2 916 231 patients from 32 cohort studies were included in the quantitative and qualitative synthesis. The meta-analysis showed that metformin was significantly associated with lower mortality in COVID-19 patients with DM in both unadjusted (OR 0.61 [95% confidence interval: 0.53-0.71], P < .00001, I2  = 70%) and adjusted (OR 0.78 [95% confidence interval: 0.69-0.88], P < .00001, I2  = 67%) models. CONCLUSION: Poor outcomes in COVID-19 patients with DM can be attributed to inadequate glycaemic control and weakened immune responses. Metformin has multiple effects that can improve outcomes in patients with DM and our findings highlight a possible role of its use. However, robust randomised trials are needed to thoroughly assess its use.

Sex differences in the role of phospholipase A2 -dependent arachidonic acid pathway in the perivascular adipose tissue function in pigs.

KEY POINTS: The fat surrounding blood vessels (perivascular adipose tissue or PVAT) releases vasoactive compounds that regulate vascular smooth muscle tone. There are sex differences in the regulation of vascular tone, but, to date, no study has investigated whether there are sex differences in the regulation of blood vessel tone by PVAT. This study has identified that the cyclooxygenase products thromboxane and PGF2α are released from coronary artery PVAT from pigs. Thromboxane appears to mediate the PVAT-induced contraction in arteries from females, whereas PGF2α appears to mediate the contraction in arteries from males. These sex differences in the role of these prostanoids in the PVAT-induced contraction can be explained by a greater release of thromboxane from PVAT from female animals and greater sensitivity to PGF2α in the porcine coronary artery from males. ABSTRACT: Previous studies have demonstrated that perivascular adipose tissue (PVAT) causes vasoconstriction. In this present study, we determined the role of cyclooxygenase-derived prostanoids in this contractile response and determined whether there were any sex differences in the regulation of vascular tone by PVAT. Contractions in isolated segments of coronary arteries were determined using isolated tissue baths and isometric tension recording. Segments were initially cleaned of PVAT, which was then re-added to the tissue bath and changes in tone measured over 1 h. Levels of PGF2α and thromboxane B2 (TXB2 ) were quantified by ELISA, and PGF2α (FP) and thromboxane A2 (TP) receptor expression determined by Western blotting. In arteries from both male and female pigs, re-addition of PVAT caused a contraction, which was partially inhibited by the cyclooxygenase inhibitors indomethacin and flurbiprofen. The FP receptor antagonist AL8810 attenuated the PVAT-induced contraction in arteries from males, whereas the TP receptor antagonist GR32191B inhibited the PVAT-induced contraction in arteries from females. Although there was no difference in PGF2α levels in PVAT between females and males, PGF2α produced a larger contraction in arteries from males, correlating with a higher FP receptor expression. In contrast, release of TXB2 from PVAT from females was greater than from males, but there was no difference in the contraction by the TXA2 agonist U46619, or TP receptor expression in arteries from different sexes. These findings demonstrate clear sex differences in PVAT function in which PGF2α and TXA2 antagonists can inhibit the PVAT-induced vasoconstriction in male and female PCAs, respectively.

Hyperoxic gassing with Tiron enhances bradykinin-induced endothelium-dependent and EDH-type relaxation through generation of hydrogen peroxide.

Oxygenation with 95%O2 is routinely used in organ bath studies. However, hyperoxia may affect tissue responses, particularly in studies which involve reactive oxygen species (ROS). Here, the effects of the antioxidant, Tiron, were investigated under different gassing conditions in the porcine isolated coronary artery (PCA). Distal PCAs from male and female pigs were mounted in a wire myograph gassed with either 95%O2/5%CO2 or 95% air/5%CO2 and pre-contracted with U46619. Concentration-response curves to bradykinin were constructed in the presence of Tiron (1mM), a cell permeable superoxide scavenger and catalase (1000Uml(-1)) to breakdown H2O2. The H2O2 level in Krebs'-Henseleit solution was detected using Amplex Red. Bradykinin produced concentration-dependent vasorelaxations in male and female PCAs when gassed with either 95%O2 or air, with no differences in the Rmax or EC50. Tiron increased the potency of bradykinin only when gassed with 95%O2 in PCAs from both sexes. At 95%O2, catalase prevented the leftward shift caused by Tiron in both sexes indicating that catalase prevented the formation of H2O2 by Tiron. In female PCAs, addition of catalase to Tiron significantly reduced the Rmax. In the EDH-type response (using L-NAME and indomethacin), Tiron enhanced the potency of the bradykinin-induced vasorelaxation when gassed with 95%O2 in PCAs from both sexes. Biochemical analysis using Amplex Red demonstrated that H2O2 was generated in Krebs'-Henseleit solution when gassed with 95%O2, but not with air. Therefore, hyperoxic gassing conditions could alter the environment generating superoxide within the Krebs'-Henseleit buffer, which may, in turn, influence the in vitro pharmacological responses.

Cannabidiol improves vasorelaxation in Zucker diabetic fatty rats through cyclooxygenase activation.

Cannabidiol (CBD) decreases insulitis, inflammation, neuropathic pain, and myocardial dysfunction in preclinical models of diabetes. We recently showed that CBD also improves vasorelaxation in the Zucker diabetic fatty (ZDF) rat, and the objective of the present study was to establish the mechanisms underlying this effect. Femoral arteries from ZDF rats and ZDF lean controls were isolated, mounted on a myograph, and incubated with CBD (10 µM) or vehicle for 2 hours. Subsequent vasorelaxant responses were measured in combination with various interventions. Prostaglandin metabolites were detected using enzyme immunoassay. Direct effects of CBD on cyclooxygenase (COX) enzyme activity were measured by oxygraph assay. CBD enhanced the maximum vasorelaxation to acetylcholine (ACh) in femoral arteries from ZDF lean rats (P < 0.01) and especially ZDF rats (P < 0.0001). In ZDF arteries, this enhancement persisted after cannabinoid receptor (CB) type 1, endothelial CB, or peroxisome proliferator-activated receptor-γ antagonism but was inhibited by CB2 receptor antagonism. CBD also uncovered a vasorelaxant response to a CB2 agonist not previously observed. The CBD-enhanced ACh response was endothelium-, nitric oxide-, and hydrogen peroxide-independent. It was, however, COX-1/2- and superoxide dismutase-dependent, and CBD enhanced the activity of both purified COX-1 and COX-2. The CBD-enhanced ACh response in the arteries was inhibited by a prostanoid EP4 receptor antagonist. Prostaglandin E2 metabolite levels were below the limits of detection, but 6-keto prostaglandin F1 α was decreased after CBD incubation. These data show that CBD exposure enhances the ability of arteries to relax via enhanced production of vasodilator COX-1/2-derived products acting at EP4 receptors.

An Integrated Dyspepsia Module for First-year Pharmacy Students.

Objective. To design an integrated dyspepsia module for first year pharmacy students that combines clinical and professional practice with fundamental sciences in five different science subject areas. Methods. The approaches used in designing this module are described with emphasis on strategies adopted to integrate science and practice, and the new ways of working adopted by the design team. Students' views and experiences of the module and its integration were explored using questionnaires. Results. A high proportion of students reported positive views and experiences of the module, the integration and its impact (as self-reported) on their learning and practice. The assessment of student performance indicated learning and attainment was at an appropriate level for a first-year module. Both the student grades and research results indicate a positive student learning experience. Conclusion. The dyspepsia module provides a flexible and effective template for the integration of science and practice in theme-based modules, with students reporting positively about the integration, including their perception of its contribution to improving their learning and understanding. New and more collaborative ways of working are required when designing integrated modules.

Sex differences in the regulation of porcine coronary artery tone by perivascular adipose tissue: a role of adiponectin?

BACKGROUND AND PURPOSE: As there is sexual dimorphism in the regulation of vascular tone, the aim of this present study was to determine whether there are sex differences in perivascular adipose tissue (PVAT)-mediated regulation of the porcine coronary artery (PCA) tone. EXPERIMENTAL APPROACH: Isometric tension recording system was used to record changes in tone in PCAs. Western blot analysis was performed to examine the expression of adiponectin in PVAT and adiponectin receptors and adiponectin binding protein (APPL1) in PCA. The level of adiponectin released from PVAT was measured using elisa. KEY RESULTS: In the presence of adherent PVAT, contractions to the thromboxane mimetic U46619 and endothelin-1 were significantly reduced in PCAs from females, but not males. In PCAs pre-contracted with U46619, re-addition of PVAT caused relaxation in PCAs from females, but not males. This relaxant response in females was attenuated by combined inhibition of NOS (with L-NAME) and COX (with indomethacin). Pre-incubation with an anti-adiponectin antibody abolished the relaxant effects of PVAT. The adiponectin receptor agonist (adipoRon) produced a greater relaxation in PCAs from females compared with males. However, there was no difference in either the expression or release of adiponectin from PVAT between sexes. Similarly, there was no difference in the expression of adiponectin receptors or the adiponectin receptor adaptor protein APPL1 in PCAs. CONCLUSION AND IMPLICATIONS: These findings demonstrate a clear sex difference in the regulation of coronary arterial tone in response to adiponectin receptor stimulation, which may underlie the anticontractile effects of PVAT in females.

In Vivo Cannabidiol Treatment Improves Endothelium-Dependent Vasorelaxation in Mesenteric Arteries of Zucker Diabetic Fatty Rats.

Background and purpose: We have shown that in vitro treatment with cannabidiol (CBD, 2 h) enhances endothelial function in arteries from Zucker diabetic fatty (ZDF) rats, partly due to a cyclooxygenase (COX)-mediated mechanism. The aim of the present study was to determine whether treatment with CBD in vivo would also enhance endothelial function. Experimental approach: Male ZDF rats, or ZDF Lean rats, were treated for 7 days (daily i.p. injection) with either 10mg/kg CBD or vehicle (n = 6 per group). Sections of mesenteric resistance arteries, femoral arteries and thoracic aortae were mounted on a wire myograph, and cumulative concentration-response curves to endothelium-dependent (acetylcholine, ACh, 1 nM-100 µM) or endothelium-independent (sodium nitroprusside, SNP, 1 nM-100 µM) agents were constructed. Multiplex analysis was used to measure serum metabolic and cardiovascular biomarkers. Key results: Vasorelaxation to ACh was significantly enhanced in mesenteric arteries from CBD-treated ZDF rats, but not ZDF Lean rats. The enhanced vasorelaxation in ZDF mesenteric arteries was no longer observed after COX inhibition using indomethacin or nitric oxide (NO) inhibition using L-NAME. Increased levels of serum c-peptide, insulin and intracellular adhesion molecule-1 observed in the ZDF compared to ZDF Lean rats were no longer significant after 7 days CBD treatment. Conclusion and implications: Short-term in vivo treatment with CBD improves ex vivo endothelium-dependent vasorelaxation in mesenteric arteries from ZDF rats due to COX- or NO-mediated mechanisms, and leads to improvements in serum biomarkers.

Differential effects of low and high dose folic acid on endothelial dysfunction in a murine model of mild hyperhomocysteinaemia.

The exact mechanism(s) by which hyperhomocysteinaemia promotes vascular disease remains unclear. Moreover, recent evidence suggests that the beneficial effect of folic acid on endothelial function is independent of homocysteine-lowering. In the present study the effect of a low (400 microg/70 kg/day) and high (5 mg/70 kg/day) dose folic acid supplement on endothelium-dependent relaxation in the isolated perfused mesenteric bed of heterozygous cystathionine beta-synthase deficient mice was investigated. Elevated total plasma homocysteine and impaired relaxation responses to methacholine were observed in heterozygous mice. In the presence of N(G)-nitro-L-arginine methyl ester relaxation responses in wild-type tissues were reduced, but in heterozygous tissues were abolished. Clotrimazole and 18alpha-glycyrrhetinic acid, both inhibitors of non-nitric oxide/non-prostanoid-induced endothelium-dependent relaxation, reduced responses to methacholine in wild-type but not heterozygous tissues. The combination of N(G)-nitro-L-arginine methyl ester and either clotrimazole or 18alpha-glycyrrhetinic acid completely inhibited relaxation responses in wild-type tissues. Both low and high dose folic acid increased plasma folate, reduced total plasma homocysteine and reversed endothelial dysfunction in heterozygous mice. A greater increase in plasma folate in the high dose group was accompanied by a more significant effect on endothelial function. In the presence of N(G)-nitro-L-arginine methyl ester, a significant residual relaxation response was evident in tissues from low and high dose folic acid treated heterozygous mice. These data suggest that the impaired mesenteric relaxation in heterozygous mice is largely due to loss of the non-nitric oxide/non-prostanoid component. While low dose folic acid may restore this response in a homocysteine-dependent manner, the higher dose has an additional effect on nitric oxide-mediated relaxation that would appear to be independent of homocysteine lowering.

Cardiovascular effects of cannabinoids.

The prototypic endocannabinoid, anandamide, and synthetic analogues have been shown to elicit pressor and depressor effects, bradycardia, vasorelaxation, and inhibition of neurotransmission in the central and peripheral nervous systems. Cannabinoid-mediated inhibition of neurotransmission is mediated by inhibition of voltage-gated Ca(2+) channels and adenylyl cyclase and activation of inwardly rectifying K(+) channels. The precise mechanisms underlying the vasorelaxant actions of cannabinoids are currently unclear, but might involve both receptor-dependent and -independent and endothelium-dependent and -independent pathways. Mechanisms proposed have included the release of endothelial autacoids, activation of myoendothelial gap junctions, activation of the Na(+) pump, activation of K(+) channels, inhibition of Ca(2+) channels, and activation of vanilloid receptors, leading to the release of sensory neurotransmitters. Pathophysiologically, the vasodilator actions of endocannabinoids have been implicated in the hypotension associated with both septic and haemorrhagic shock, but their physiological significance remains to be determined.

The effects of Delta9-tetrahydrocannabinol in rat mesenteric vasculature, and its interactions with the endocannabinoid anandamide.

1 Delta9-tetrahydrocannabinol (THC) produces varying effects in mesenteric arteries: vasorelaxation (third-order branches, G3), modest vasorelaxation (G2), no effect (G1) and vasoconstriction (the superior mesenteric artery, G0). 2 In G3, vasorelaxation to THC was inhibited by pertussis toxin, but was unaffected by the CB1 receptor antagonist, AM251 (1 microM), incubation with the TRPV1 receptor agonist capsaicin (10 microM, 1 h), the TRPV1 receptor antagonist capsazepine (10 microM) or de-endothelialisation. 3 In G3, vasorelaxation to THC was inhibited by high K+ buffer, and by the following K+ channel inhibitors: charybdotoxin (100 nM), apamin (500 nM) and barium chloride (30 microM), but not by 4-aminopyridine, glibenclamide or tertiapin. 4 In G3, THC (10 and 100 microM) inhibited the contractile response to Ca2+ in a Ca2+-free, high potassium buffer, indicating that THC blocks Ca2+ influx. 5 In G0, the vasoconstrictor responses to THC were inhibited by de-endothelialisation and SR141716A (100 nM), but not by the endothelin (ET(A)) receptor antagonist FR139317 (1 microM).THC (1 and 10 microM) antagonised vasorelaxation to anandamide in G3 but not G0. THC did not antagonise the noncannabinoid verapamil, capsaicin or the CB1 receptor agonist CP55,940. 6 THC (10 and 100 microM) inhibited endothelium-derived relaxing factor (EDHF)-mediated responses to carbachol in a manner similar to the gap junction inhibitor 18alpha-glycyrrhetinic acid. 7 These data show that THC causes vasorelaxation through activation of K+ channels and inhibition of Ca2+ channels, and this involves non-CB1, non-TRPV1 but G-protein-coupled receptors. In G0, THC does not cause relaxation and at high concentrations causes contractions. Importantly, THC antagonises the effects of anandamide, possibly through inhibition of EDHF activity.

Vascular effects of delta 9-tetrahydrocannabinol (THC), anandamide and N-arachidonoyldopamine (NADA) in the rat isolated aorta.

The vascular effects of cannabinoids have been compared in the rat isolated aorta. Delta9-Tetrahydrocannabinol (THC), anandamide and N-arachidonoyl-dopamine (NADA) all caused vasorelaxation to similar degrees in pre-constricted aortae. Vasorelaxation to THC was inhibited by in vivo pre-treatment with pertussis toxin (10 microg/kg) or with the synthetic cannabinoid CP55,940 (((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol), acutely or chronically), exposure to capsaicin in vitro (10 microM for 1 h), and de-endothelialisation. Vasorelaxation to anandamide was only inhibited by pertussis toxin and chronic CP55,940 pre-treatment (0.4 mg/kg for 11 days). Vasorelaxation to NADA was inhibited by pertussis toxin and chronic CP55,940 pre-treatment, and by de-endothelialisation. The vasorelaxant effects of the cannabinoids were not inhibited by cannabinoid CB1 receptor antagonism; however, vasorelaxation to both CP55,940 and THC was inhibited by cannabinoid CB2 receptor antagonism. Vasorelaxation to all cannabinoids was enhanced in the presence of indomethacin (10 microM). THC also caused vasoconstriction of the aorta while anandamide, NADA, CP55,940 and WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4benzoxazin-yl]-(1-naphthalenyl)methanone mesylate) did not. The vasoconstrictor effects of THC were inhibited by in vivo pre-treatment with pertussis toxin or CP55,940, acute exposure to CP55,940, cannabinoid CB1 receptor antagonism and cyclooxygenase inhibition. These results demonstrate the opposing vascular effects of cannabinoids in the rat aorta, and although vasorelaxation to each of the cannabinoids is of similar magnitude, it is mediated through different pathways. This gives further indication of the different vascular actions of cannabinoid compounds.

Sex differences in the role of NADPH oxidases in endothelium-dependent vasorelaxation in porcine isolated coronary arteries.

The present study examined whether vascular function, expression and activity of NADPH oxidases differ between sexes in porcine isolated coronary arteries (PCAs) using selective Nox inhibitors, ML-171 and VAS2870. Vascular responses of distal PCAs were examined under myographic conditions in the presence of a range of inhibitors. Nox activity in PCA homogenates was assessed using lucigenin-enhanced chemiluminescence. Protein expression of Nox1, Nox2 and Nox4 was compared using Western immunoblotting. The presence of ML-171 or DPI had no effect on the bradykinin-induced vasorelaxation in PCAs from females. In males, DPI shifted the EC50 2.8-fold to the right. In the presence of L-NAME and indomethacin, DPI and ML-171 had no effect in females, but enhanced the bradykinin-induced vasorelaxation in males. ML-171 had no effect on the forskolin-induced vasorelaxation but decreased the potency of U46619-induced tone in both sexes in the absence or presence of endothelium. VAS2870 had no effect on the bradykinin-induced vasorelaxation in both sexes but reduces the EDH-type response in males only. Nox activity was reduced by DPI and ML-171, but not VAS2870 in PCAs from both sexes. Protein expression of Nox1 and Nox2 in PCAs was higher in males compared to females whereas Nox4 was higher in females. Inhibition of Nox with ML-171 enhances while VAS2870 reduces the EDH-type response in PCAs from males but not females. This indicates that Nox-generated ROS play a role in the EDH-type response in males with differences attributed to the differential expression of Nox isoforms. This may underlie the greater oxidative stress observed in males.

Sex differences in the role of transient receptor potential (TRP) channels in endothelium-dependent vasorelaxation in porcine isolated coronary arteries.

Endothelial and smooth muscle Transient Receptor Potential (TRP) channels contribute to regulation of vascular tone. We have previously reported sex differences in the endothelial function in porcine isolated coronary arteries (PCAs). The present study examined the role of TRP channels in endothelium-dependent and H2O2-induced vasorelaxations in male and female PCAs. Distal PCAs were mounted in a wire myograph and precontracted with U46619. Concentration-response curves to bradykinin, H2O2 and A23187 were constructed in the presence of TRP channel antagonists with or without L-NAME and indomethacin to inhibit NO synthase and cyclooxygenase respectively. 2-APB (TRPC & TRPM antagonist) inhibited the maximum relaxation (Rmax) of the bradykinin-induced vasorelaxation and abolished the EDH-type response in PCAs from both sexes. SKF96365 (TRPC antagonist) inhibited the Rmax of bradykinin-induced vasorelaxation in males, and inhibited Rmax of the EDH-type response in both sexes. Pyr3 (TRPC3 antagonist) inhibited both the NO and EDH components of the bradykinin-induced vasorelaxation in males, but not females. RN1734 (TRPV4 antagonist) reduced the potency of the NO component of the bradykinin-induced vasorelaxation in females only, but inhibited the Rmax of the EDH-type component in both sexes. 2-APB, SKF96365 and RN1734 all reduced the H2O2-induced vasorelaxation, whereas Pyr3 had no effect. No differences in expression level of TRPC3 and TRPV4 between sexes were detected using Western blot. Present study demonstrated a clear sex differences in the role TRP channels where TRPC3 play a role in the NO- and EDH-type response in males and TRPV4 play a role in the NO-mediated response in females.

Testosterone-induced vasorelaxation in the rat mesenteric arterial bed is mediated predominantly via potassium channels.

We have investigated the involvement of nitric oxide and K(+) channels in the vasorelaxant responses to physiologically-relevant concentrations of testosterone in the rat isolated mesenteric arterial bed. Testosterone (100 pM - 10 microM) elicited concentration-dependent relaxations in the isolated mesenteric arterial bed (pEC(50)=9.47 (9.22 - 9.73, 95% CI), maximal relaxation, R(max)=62.8+/-2.0%, n=6). A nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 300 microM) or removal of the endothelium significantly inhibited maximal relaxations to testosterone (L-NAME: R(max)=51.4+/-1.1%, P<0.01, n=6; endothelium-denuded: R(max)=46.9+/-2.8%, P<0.001, n=5). Raising the extracellular K(+) concentration to 30 and 60 mM, or pre-treatment with 300 microM tetrabutylammonium chloride (TBA), a calcium-activated K(+) channel inhibitor, abolished vasorelaxations induced by testosterone. A selective inhibitor of ATP-sensitive K(+) (K(ATP)) channels, glibenclamide (10 microM) and an inhibitor of voltage-sensitive K(+) (K(V)) channels, 4-aminopyridine (4-AP, 1 mM) did not affect testosterone-induced responses. Vasorelaxation to 1 microM testosterone was significantly (P<0.05) inhibited by 100 nM charybdotoxin (ChTx), an inhibitor of large conductance calcium-activated K(+) (BK(Ca)) channels (control: 63.3+/-9.9%, n=6; ChTx: 11.9+/-12.7%, n=3). Neither the testosterone receptor antagonist, flutamide (10 microM) nor an aromatase inhibitor, aminoglutethimide (10 microM) inhibited testosterone-induced responses. In conclusion, the present findings demonstrate, in the rat isolated mesenteric arterial bed, that testosterone causes acute vasorelaxations at physiologically relevant concentrations which are, in part, mediated via NO- and endothelium-dependent pathways. However, the activation of BK(Ca) channels plays a substantial role in testosterone-induced vasorelaxation.

Characterisation of the vasorelaxant properties of the novel endocannabinoid N-arachidonoyl-dopamine (NADA).

1. We have investigated the vascular effects of N-arachidonoyl-dopamine (NADA), a novel endocannabinoid/vanilloid. NADA caused vasorelaxant effects comparable to those of anandamide in small mesenteric vessels (G3), the superior mesenteric artery (G0) and in the aorta. 2. In G3, addition of N(G)-nitro-l-arginine methyl ester (300 microm) or the dopamine (D(1)) receptor antagonist (SCH23390, 1 microm) did not affect responses to NADA. In the presence of 60 mm KCl, after de-endothelialisation, or after K(+) channel inhibition with charybdotoxin (100 nm) and apamin (500 nm), relaxant responses to NADA were inhibited. 3. In G3, pretreatment with the vanilloid receptor (VR) agonist capsaicin (10 microm) or the VR antagonist capsazepine (10 microm) reduced vasorelaxation to NADA. 4. In G3, application of the CB(1) antagonist SR141716A at 1 microm but not 100 nm reduced the potency of NADA. Another CB(1) antagonist, AM251 (100 nm and 1 microm), did not affect vasorelaxation to NADA. After endothelial denudation, SR141716A (1 microm) did not reduce the responses further. A combination of capsaicin and SR141716A (1 microm) reduced vasorelaxation to NADA further than with capsaicin pretreatment alone. The novel endothelial cannabinoid (CB) receptor antagonist O-1918 opposed vasorelaxation to NADA in G3. 5. In the superior mesenteric artery (G0), vasorelaxation to NADA was not dependent on an intact endothelium and was not sensitive to O-1918, but was sensitive to capsaicin and SR141716A or AM251 (both 100 nm). 6. The results of the present study demonstrate for the first time that NADA is a potent vasorelaxant. In G3, the effects of NADA are mediated by stimulation of the VR and the novel endothelial CB receptor, while in G0, vasorelaxation is mediated through VR(1) and CB(1) receptors.

The complexities of the cardiovascular actions of cannabinoids.

The cardiovascular actions of cannbinoids are complex. In general they cause vasorelaxation in isolated blood vessels, while in anaesthetised animals they cause multiphasic responses which involve an early bradycardia and long-lasting hypotension. However, in conscious animals, the picture is one of bradycardia followed by pressor responses. Clearly, the responses to cannabinoids are dependent on the experimental conditions and synthetic cannabinoids and endocannabinoids exhibit different pharmacologies. In terms of mechanisms involved in the vascular responses to cannabinoids, the following have been implicated: the involvement of 'classical' cannabinoid receptors, the involvement of a novel endothelial cannabinoid receptor, the release of nitric oxide, the release of endothelium-derived hyperpolarising factor (EDHF), the activation of vanilloid receptors, metabolism of endocannabinoids to vasoactive molecules, and both peripheral inhibition and central excitation of the sympathetic nervous system.

Heterogeneity in the mechanisms of vasorelaxation to anandamide in resistance and conduit rat mesenteric arteries.

1 In order to address mechanistic differences between arterial vessel types, we have compared the vasorelaxant actions of anandamide in resistance (G3) and conduit (G0) mesenteric arteries. 2 Anandamide produced concentration-dependent relaxations of pre-constricted G3 arteries with a maximal response that was significantly greater than seen in G0. 3 The CB1 receptor selective antagonists SR141716A (100 nm) and AM251 (100 nm) caused reductions in the vasorelaxant responses to anandamide in both arteries. Maximal vasorelaxant responses to anandamide were reduced in both arteries after treatment with capsaicin to deplete sensory neurotransmitters (10 microm for 1 h). 4 Vasorelaxation to anandamide was not affected by the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 300 microm) in either artery. Only responses in G3 arteries were sensitive to removal of the endothelium. In G3 vessels only, vasorelaxation to anandamide was reduced by inhibition of EDHF activity with a combination of charybdotoxin (100 nm) and apamin (500 nm) in the presence of L-NAME (300 microm) and indomethacin (10 microm). 5 Antagonism of the novel endothelial cannabinoid receptor (O-1918, 1 microm) caused a reduction in the sensitivity to anandamide in G3 but not G0. 6 G3, but not G0, vessels showed a small reduction in vasorelaxant responses to anandamide after inhibition of gap junctional communication with 18alpha-GA (100 microm). 7 These results demonstrate that there are differences in the mechanisms of vasorelaxation to anandamide between conduit and resistance mesenteric arteries. In small resistance vessels, vasorelaxation occurs through stimulation of vanilloid receptors, CB1 receptors, and an endothelial receptor coupled to EDHF release. By contrast, in the larger mesenteric artery, vasorelaxation is almost entirely due to stimulation of vanilloid receptors and CB1 receptors, and is endothelium-independent.

The role of gap junctions in mediating endothelium-dependent responses to bradykinin in myometrial small arteries isolated from pregnant women.

Endothelium-dependent responses were assessed in myometrial small arteries isolated from pregnant women, using pressure myography. Responses to bradykinin were unaffected by the combined presence of the nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME, 100 micro M) and the cyclo-oxygenase inhibitor, indomethacin (10 micro M). The additional presence of clotrimazole (50 micro M) attenuated, but did not abolish, vasodilator responses to bradykinin. Raising extracellular [K(+)] (by between 1 and 15 mM) did not evoke a vasodilator response, nor did the cannabinoids, anandamide and methanandamide. Responses to bradykinin were attenuated in the presence of the gap junction inhibitors 18-alpha-glycyrrhetinic acid (18-alpha GA, 100 micro M), carbenoxolone (100 micro M) and palmitoleic acid (50 micro M). SR141716A, the CB(1) receptor antagonist attenuated responses to bradykinin, but only at high concentrations (10 micro M). These results suggest that gap junctional communication is involved in the nitric oxide (NO)- and prostanoid-independent vasodilator responses to bradykinin in myometrial small arteries in normal pregnancy.

Mechanisms of vasorelaxation to testosterone in the rat aorta.

We have investigated the role of endothelium-derived relaxing factors, K(+) channels and steroid receptors in vasorelaxation to testosterone in the rat aorta. Testosterone (1 nM-mM) caused acute concentration-dependent vasorelaxation. Both indomethacin (10 microM) and flurbiprofen (10 microM) uncovered relaxant responses to testosterone. The action of indomethacin was inhibited by endothelial removal. N(G)-nitro-L-arginine methyl ester (L-NAME, 300 microM) had no effects on testosterone-induced responses. In the presence of indomethacin, the vasorelaxant potency of testosterone was reduced by depolarization with 60 mM KCl or charybdotoxin (100 nM), but not by glibenclamide (10 microM), 4-aminopyridine (1 mM) or barium chloride (30 microM). The responses to testosterone were not inhibited by flutamide (10 microM) or mifepristone (30 microM). Pre-treatment of the aorta with testosterone (100 microM) inhibited CaCl(2)-induced contraction. In the present study, we have demonstrated that testosterone causes acute vasorelaxations, which are modulated via endothelium-derived prostanoids. The responses uncovered by cyclooxygenase inhibitors are due to the activation of K(Ca) channels, while at higher concentrations, testosterone inhibits Ca(2+) influx.

Mechanisms of vasorelaxation to 17beta-oestradiol in rat arteries.

We have investigated the involvement of the endothelium, K+ channels, oestradiol receptors, and Ca2+ influx in 17beta-oestradiol-induced vasorelaxation in rat mesenteric arterial beds and aortae. 17beta-Oestradiol (10 pM-1 mM) caused acute vasorelaxations in mesenteric arterial beds and aortae from male and female rats. In male rat mesenteric vessels and aortae, the vasorelaxations were mostly independent of the endothelium and nitric oxide (NO). However, indomethacin (10 microM) enhanced the relaxant responses to 17beta-oestradiol. In male rat mesenteric beds, 60 mM KCl, tetrabutylammonium chloride (300 microM), 4-aminopyridine (1 mM), and barium chloride (30 microM), charybdotoxin (100 nM), but not glibenclamide (10 microM) and tamoxifen (10 microM), inhibited vasorelaxation to 17beta-oestradiol. In male rat aortae, 60 mM KCl did not affect vasorelaxation to 17beta-oestradiol. However, in the presence of indomethacin, vasorelaxation to 17beta-oestradiol was enhanced but this was sensitive to 60 mM KCl. Pre-treatment with 17beta-oestradiol (100 microM) inhibited CaCl2-induced contraction. The present findings indicate that, in rat mesenteric beds and aortae, 17beta-oestradiol causes acute and potent vasorelaxation which may be enhanced in the presence of a cyclooxygenase inhibitor. In mesenteric arterial bed, 17beta-oestradiol-induced vasorelaxation occurs primarily via activation of K+ channels. In the aorta, vasorelaxations involved activation of K+ efflux when the cyclooxygenase pathway was inhibited, and also inhibition of Ca2+ influx.