Paula J. W. Smith

Anandamide induces cardiovascular and respiratory reflexes via vasosensory nerves in the anaesthetized rat

We tested the hypothesis that sensory nerves innervating blood vessels play a role in the local and systemic regulation of the cardiovascular and respiratory (CVR) systems. We measured CVR reflexes evoked by administration of anandamide (86 – 863 nmoles) and capsaicin (0.3 – 10 nmoles) into the hindlimb vasculature of anaesthetized rats. Anandamide and capsaicin each caused a rapid dose‐dependent reflex fall in blood pressure and an increase in ventilation when injected intra‐arterially into the hindlimb. Action of both agonists at the vanilloid receptor (VR1) on perivascular sensory nerves was investigated using capsazepine (1 mg kg−1 i.a.) a competitive VR1 antagonist, ruthenium red (1 mg kg−1 i.a.), a non‐competitive antagonist at VR1, or a desensitizing dose of capsaicin (200 nmoles i.a.). The cannabinoid receptor antagonist SR141716 (1 mg kg−1 i.a.) was used to determine agonist activity at the CB1 receptor. Capsazepine, ruthenium red, or acute VR1 desensitization by capsaicin‐pretreatment, markedly attenuated the reflex CVR responses evoked by anandamide and capsaicin (P<0.05; paired Students t‐test). Blockade of CB1 had no significant effect on the responses to anandamide. Local sectioning of the femoral and sciatic nerves attenuated CVR responses to anandamide and capsaicin (P<0.05). Vagotomy or carotid sinus sectioning had no significant effect on anandamide‐ or capsaicin‐induced responses. These data demonstrate that both the endogenous cannabinoid, anandamide, and the vanilloid, capsaicin, evoke CVR reflexes when injected intra‐arterially into the rat hindlimb. These responses appear to be mediated reflexly via VR1 located on sensory nerve endings within the hindlimb vasculature.

Coordinated upregulation of the cardiac endothelin system in a rat model of heart failure

The potent vascular, cardiac, and renal actions of endothelin-1 (ET-1) suggest a role for this vasoconstrictor peptide in the pathophysiology of heart failure (HF). Recent studies have shown increased levels of ET-1 peptide accompanied by increased ETB receptor binding in the left ventricle during experimental HF. However, much less is known about the regulation of mRNA expression of these genes in HF. We compared the levels of mRNA expression for ET-1 and ET receptors (ETA and ETB) in the left ventricle of rats with HF induced by coronary artery ligation (n = 6) vs. sham-operated animals (n = 6). Levels of mRNA for ET-1 were determined by ribonuclease protection assay (RPA) using beta-actin as the internal control, whereas ET receptors were quantified by quantitative-competitive RT-PCR. Compared with sham animals, ET-1, ETA, and ETB receptor mRNA levels were markedly upregulated in the left ventricle by 6.6 +/- 1.8-fold (p < 0.01), 3.2 +/- 0.6-fold (p < 0.05), and 3.5 +/- 1.0-fold (p < 0.05), respectively. ET-1 mRNA levels were measured in two additional groups of rats (HF and sham; n = 6 each) treated for 4 weeks with the selective ETA receptor antagonist LU135252. This treatment had no significant effect on ET-1 mRNA expression in sham animals but reduced the upregulation of ET-1 expression in the HF group by 41 +/- 19% (p < 0.05). This study confirms the potential importance of ET-1 in HF and suggests that increased expression of ET-1 and ET receptors in the failing ventricle may contribute to alteration in basal cardiac contractility and myocardial remodeling.

Expression of Cannabinoid CB1 Receptors in Models of Diabetic Neuropathy

A clearer understanding of the mechanisms underlying the development and progression of diabetic neuropathy is likely to indicate new directions for the treatment of this complication of diabetes. In the present study we investigated the expression of cannabinoid CB1 receptors in models of diabetic neuropathy. PC12 cells were differentiated into a neuronal phenotype with nerve growth factor (NGF) (50 ng/ml) in varying concentrations of glucose (5.5–50 mM). CB1 receptor expression was studied at the mRNA level by reverse transcriptase-polymerase chain reaction (RT-PCR) and at the protein level via immunohistochemical and Western blot analysis. CB1 expression was also compared in dorsal root ganglia (DRG) removed from streptozotocin-induced diabetic rats versus control animals. Total neurite length induced by NGF was reduced in cells cultured in 20 to 50 mM glucose at day 6 (P < 0.01 versus 5.5 mM; n = 6). Cell viability assays conducted in parallel on day 6 confirmed that the total cell numbers were not significantly different among the various glucose concentrations (P = 0.86; n = 12). RT-PCR, immunohistochemical, and Western blot analysis all revealed down-regulation of the CB1 receptor in cells treated with high glucose (P < 0.05; n = 4–5 for each), and in DRG removed from diabetic rats compared with controls (P < 0.01; n = 5 for immunohistochemistry, and n = 3 for Western blot). These results suggest that high glucose concentrations are associated with decreased expression of CB1 receptors in nerve cells. Given the neuroprotective effect of cannabinoids, a decline in CB1 receptor expression may contribute to the neurodegenerative process observed in diabetes.

Expression of Endothelial Factors After Arterial Injury in the Rat

Endothelin-1 (ET-1) and nitric oxide (NO) are potent vasoactive factors known to play a role in vascular remodeling. This study assessed the temporal expression of endothelial NO synthase (eNOS), preproET-1, and ETA and ETB receptor mRNAs in the rat carotid artery after balloon injury using quantitative competitive reverse transcription-polymerase chain reaction (qcRT-PCR) and the ribonuclease protection assay (RPA). Levels of ET-1 increased sharply after arterial injury, peaking (5.1-fold) at 2 days. This was associated with a dramatic increase in the expression of ETB (63-fold) and ETA (158-fold) receptor mRNA, peaking at days 1 and 2, respectively. Expression of eNOS was not detectable immediately after balloon injury, consistent with complete denudation, but reappeared after day 2 and increased to preinjury levels by day 14. The recovery of eNOS expression mirrored the return of ET-1 and ET receptor expression to baseline levels. The results confirm profound upregulation of the ET system in this model of arterial injury and suggest a critical role for eNOS expression and re-endothelialization in the normalization of ET-1 and ET receptor expression during the recovery phase, events that may be important in long-term arterial patency.

Regulation of endothelin-B receptor mRNA expression in human endothelial cells by cytokines and growth factors.

The regulation of endothelin-B receptor (ETB) mRNA expression in human endothelial cells (ECs) by cytokines and growth factors may play an important role in the response of the endothelium to inflammatory and angiogenic stimuli. Using quantitative RT-PCR, we studied ETB expression in human umbilical vein ECs (HUVECs) grown in culture on either plastic or fibrin matrix for 24 h in the presence or absence of tumor necrosis factor-alpha (TNF-alpha, 100 U/ml) or basic fibroblast growth factor (bFGF, 30 ng/ml). In addition, the effect of the nitric oxide (NO) donor S-nitrosyl-acetylpenicillamine (SNAP, 0.4 mM) was examined directly on ETB expression or on the response to bFGF. Under control conditions, ETB mRNA was detected after 35 cycles of amplification as a band of the expected size (553 bp). In the absence of fibrin matrix, ETB was downregulated by bFGF and TNF-alpha and could barely be detected by PCR. Southern analysis of the RT-PCR products after 25 cycles revealed that bFGF reduced ETB mRNA expression by 2.7 +/- 0.4-fold (p < 0.01) and TNF-alpha tended to reduce its expression by 1.8 +/- 0.9-fold of control, although this did not reach statistical significance (p < 0.20). In contrast, on fibrin matrix both bFGF and TNF-alpha increased ETB mRNA expression by 25 +/- 9-fold (p < 0.05) and 68 +/- 19-fold (p < 0.05) of control, respectively, suggesting a role for ETB in the vascular tube formation that occurs under these conditions. Pharmacologic addition of NO mimicked the effect of fibrin, converting the response to bFGF from down- to upregulation of ETB, raising the possibility that NO acts as a molecular switch modulating the response to angiogenic factors.

Functional studies in small arteries do not support a primary role for endothelin in the pathogenesis of Raynaud's disease

Endothelin-1 (ET-1) has been implicated in the pathogenesis of Raynauds disease (RD). This study examined the effect of cooling on the response to ET-1 in human microvessels. Subcutaneous small arteries were dissected from gluteal fat biopsies taken from patients with RD (n = 20) and from age- and sex-matched control subjects (n = 17) and were cannulated in a small vessel arteriograph. Cumulative concentration-response curves to ET-1 (10(-12) to 3 x 10(-7) M) were obtained in vessels at 37 degrees C and 24 degrees C, with the endothelium either intact or removed (n = 6 per group). There were no significant differences in responses to ET-1 between RD patients and controls in either intact or denuded vessels, at either 37 degrees C or at 24 degrees C. There was, however, a significant endothelium-dependent interaction between the groups when the effect of temperature on the response to ET-1 was examined (p = 0.01; two-way ANOVA). Whereas cooling tended to reduce the sensitivity in RD, the opposite effect was observed in controls. Measurements of plasma ET-1 did not reveal any significant difference between patients with RD and healthy controls. These results suggest that ET-1 does not play a primary pathophysiologic role in RD. ET-1 might be responsible for mediating the prolonged vasospasm in RD, but secondary to another factor(s), such as impaired endothelium-dependent vasodilation.

The effect of cooling on the contractile response to endothelin-1 in small arteries from humans

Summary: The pathogenesis of the prolonged cold-induced vasospasm of Raynauds disease (RD) is unknown. Cooling has been shown to decrease the sensitivity of cutaneous rabbit ear arteries to endothelin-1 (ET-1), perhaps by increasing the availability of nitric oxide (NO). If the endothelium in RD lacks this NO-mediated inhibitory function during cooling, increased production and enhanced constriction to ET-1 could cause vasospasm. This study examined the effect of cooling on the contractile response to ET-1 in human microvessels. Small arteries were dissected from biopsy specimens of human fat and were cannulated in a small vessel arteriograph. Cumulative concentration-response curves to ET-1 (10 -12 to 3 × 10-7 M) were obtained in vessels at 37°C (n = 8) and 24°C (n = 7). Cooling to 24°C resulted in an eightfold decrease in sensitivity to ET-1 (EC50 6.6 ± 2.5 × 10-10 M at 37°C vs. 5.5 ± 2.5 × 10-9 M at 24°C; p < 0.05, unpaired t test). The Emax was not significantly different at 37°C and 24°C (114 ± 9 at 37°C vs. 138 ± 14 at 24°C; p = 0.18). These results suggest that cooling decreases the sensitivity of human microvessels to ET-1. Further investigation is needed to establish the mechanism of this effect, including studies in vessels obtained from patients with RD.

Selective blockade of M2 and M3 muscarinic receptors by hexahydrobenzyl-fourdapine and a comparison with zamifenacin.

1 4‐Diphenylacetoxy‐N‐cyclohexylmethyl‐piperidine HC1 (hexahydro‐benz‐4DAP) is more active as an antagonist of carbachol at receptors in guinea‐pig isolated ileum, log K (pA2) = 6.64±0.14 (s.e. 7 results), than at receptors in guinea‐pig isolated atria, log K= 5.43 ±0.14 (7). In the presence of neostigmine bromide (0.2 μm) the value for atria was 5.62 ±0.19 (4), so the lower activity on atria cannot be attributed to hydrolysis of the compound by cholinesterases present in this tissue. 2 The limit of solubility of the free base in Krebs solution (pH 7.6) is about 50 μm for both hexahydro‐benz‐4DAP and benzyl‐fourdapine (benz‐4DAP). 3 In experiments on guinea‐pig isolated ileum with hexahydro‐benz‐4DAP given together with 4‐DAMP methobromide, the combined dose‐ratio was consistent with competition: similar results were obtained with benz‐4DAP. 4 In rats anaesthetized with pentobarbitone, hexahydro‐benz‐4DAP antagonized the effects of bethanechol on blood‐pressure in doses that had little effect on heart rate or airflow. There was a limit to the effect which could be obtained, however, and the slopes of the Schild plots were less than one. The effects on rat blood‐pressure had a half‐life of at least 30 min. 5 In similar experiments with zamifenacin the slopes of the Schild plots were close to 1 and the compound was 10 to 20 times as active on blood‐pressure at it was on heart‐rate. 6 The limited solubility of the base probably accounts for the flat Schild plots obtained with hexahydro‐benz‐4DAP, which had about 10 fold selectivity for effects on blood‐pressure and was more active than expected from tests on isolated ileum.