Adrian Gericke

Adenosine Restores Angiotensin II–Induced Contractions by Receptor-Independent Enhancement of Calcium Sensitivity in Renal Arterioles

Adenosine is coupled to energy metabolism and regulates tissue blood flow by modulating vascular resistance. In this study, we investigated isolated, perfused afferent arterioles of mice, which were subjected to desensitization during repeated applications of angiotensin II. Exogenously applied adenosine restores angiotensin II–induced contractions by increasing calcium sensitivity of the arterioles, along with augmented phosphorylation of the regulatory unit of the myosin light chain. Adenosine restores angiotensin II–induced contractions via intracellular action, because inhibition of adenosine receptors do not prevent restoration, but inhibition of NBTI sensitive adenosine transporters does. Restoration was prevented by inhibition of Rho-kinase, protein kinase C, and the p38 mitogen-activated protein kinase, which modulate myosin light chain phosphorylation and thus calcium sensitivity in the smooth muscle. Furthermore, adenosine application increased the intracellular ATP concentration in LuciHEK cells. The results of the study suggest that restoration of the angiotensin II–induced contraction by adenosine is attributable to the increase of the calcium sensitivity by phosphorylation of the myosin light chain. This can be an important component of vascular control during ischemic and hypoxic conditions. Additionally, this mechanism may contribute to the mediation of the tubuloglomerular feedback by adenosine in the juxtaglomerular apparatus of the kidney.

Uncoupling of Endothelial Nitric Oxide Synthase in Perivascular Adipose Tissue of Diet-Induced Obese Mice

Objective— The present study was conducted to investigate the contribution of perivascular adipose tissue (PVAT) to vascular dysfunction in a mouse model of diet-induced obesity. Approach and Results— Obesity was induced in male C57BL/6J mice with a high-fat diet for 20 weeks, and vascular function was studied with myograph. In PVAT-free aortas isolated from obese mice, the endothelium-dependent, nitric oxide–mediated vasodilator response to acetylcholine remained normal. In contrast, a clear reduction in the vasodilator response to acetylcholine was observed in aortas from obese mice when PVAT was left in place. Adipocytes in PVAT were clearly positive in endothelial nitric oxide synthase (eNOS) staining, and PVAT nitric oxide production was significantly reduced in obese mice. High-fat diet had no effect on eNOS expression but led to eNOS uncoupling, evidenced by diminished superoxide production in PVAT after eNOS inhibition. As mechanisms for eNOS uncoupling, arginase induction and L-arginine deficiency were observed in PVAT. Obesity-induced vascular dysfunction could be reversed by ex vivo L-arginine treatment and arginase inhibition. Conclusions— Diet-induced obesity leads to L-arginine deficiency and eNOS uncoupling in PVAT. The combination therapy with L-arginine and arginase inhibitors may represent a novel therapeutic strategy for obesity-induced vascular disease.

Intraocular pressure during corneal flap preparation: comparison among four femtosecond lasers in porcine eyes.

PURPOSE To compare the course of intraocular pressure (IOP) during corneal flap preparation using four different femtosecond lasers in porcine globes. METHODS Forty-eight (12 in each group) enucleated globes were successfully cannulated through the optic nerve. Intraocular pressure was measured continuously through the cannula during a normal lamellar flap creation (regular procedure) using four femtosecond lasers (IntraLase, Abbott Medical Optics; VisuMax, Carl Zeiss Meditec AG; Femtec, Technolas Perfect Vision; and Femto LDV, Ziemer Ophthalmic Systems AG). In an additional measurement (worst-case procedure), the patient interface was pressed against the globe with increasing force until the applanation maneuver was automatically aborted by those devices capable of doing so. RESULTS During the regular procedure, the maximum IOP reached was 135±16 mmHg when using the Intra-Lase, 65±20 mmHg with the VisuMax, 205±32 mmHg with the Femtec, and 184±28 mmHg with the Femto LDV. During the worst-case procedure, a maximum IOP of 260±53 mmHg was reached with the IntraLase, 105±13 mmHg with the VisuMax, and 248±51 mmHg with the Femtec. CONCLUSIONS There is considerable variation in IOP among the tested femtosecond lasers during a regular lamellar flap creation and during the worst-case procedure. The VisuMax femtosecond laser seems to cause the lowest IOP rise in both settings.

Role of M1, M3, and M5 muscarinic acetylcholine receptors in cholinergic dilation of small arteries studied with gene-targeted mice.

Acetylcholine regulates perfusion of numerous organs via changes in local blood flow involving muscarinic receptor-induced release of vasorelaxing agents from the endothelium. The purpose of the present study was to determine the role of M₁, M₃, and M₅ muscarinic acetylcholine receptors in vasodilation of small arteries using gene-targeted mice deficient in either of the three receptor subtypes (M1R(-/-), M3R(-/-), or M5R(-/-) mice, respectively). Muscarinic receptor gene expression was determined in murine cutaneous, skeletal muscle, and renal interlobar arteries using real-time PCR. Moreover, respective arteries from M1R(-/-), M3R(-/-), M5R(-/-), and wild-type mice were isolated, cannulated with micropipettes, and pressurized. Luminal diameter was measured using video microscopy. mRNA for all five muscarinic receptor subtypes was detected in all three vascular preparations from wild-type mice. However, M(3) receptor mRNA was found to be most abundant. Acetylcholine produced dose-dependent dilation in all three vascular preparations from M1R(-/-), M5R(-/-), and wild-type mice. In contrast, cholinergic dilation was virtually abolished in arteries from M3R(-/-) mice. Deletion of either M₁, M₃, or M₅ receptor genes did not affect responses to nonmuscarinic vasodilators, such as substance P and nitroprusside. These findings provide the first direct evidence that M₃ receptors mediate cholinergic vasodilation in cutaneous, skeletal muscle, and renal interlobar arteries. In contrast, neither M₁ nor M₅ receptors appear to be involved in cholinergic responses of the three vascular preparations tested.

Cardiovascular and ocular safety of α1-adrenoceptor antagonists in the treatment of male lower urinary tract symptoms

Introduction: α1-Adrenoceptor antagonists (α-blockers) represent first-line drug treatment for male lower urinary tract symptoms. Their adverse events (AEs) include asthenia, dizziness, nasal congestion, arterial (orthostatic) hypotension and intraoperative floppy iris syndrome (IFIS). Areas covered: This report focuses on cardiovascular and ocular AEs of α-blockers as related to their mechanism of action and subtype selectivity. Expert opinion: The incidence of hypotension differs between α-blockers. It is greatest with doxazosin or terazosin, but others including tamsulosin can also lead to hypotension especially upon treatment initiation. Concomitant antihypertensive medication increases the incidence of hypotension with some α-blockers. Use of α1A-selective blockers, evening dosing and drug intake after a meal can reduce the risk of hypotension. IFIS can occur with all drugs exerting α1-adrenoceptor antagonist properties and has especially been reported for tamsulosin. It makes cataract surgery more challenging but does not constitute a health risk to patients. IFIS seems to result from inhibition of iris dilator muscle contraction and occurs in men or women, even after α-blockers have been discontinued. To reduce the risk of IFIS, the authors suggest taking a careful drug history, postponing α-blocker treatment for patients with scheduled cataract surgery and careful counseling of patients taking α-blockers.

Three commercial antibodies against α1-adrenergic receptor subtypes lack specificity in paraffin-embedded sections of murine tissues.

We tested the specificity of three commercially available antibodies (AB) against individual α1-adrenergic receptor subtypes (α1-ARST). We used these ABs to localize the α1-ARST proteins by immunohistochemistry in paraffin-embedded murine tissues. The specificity of the ABs was tested by comparing staining patterns in tissues from wild-type mice with those in corresponding tissues from mice with gene-targeted disruption of the respective α1-ARST, one of the most rigorous negative controls. None of the tested ABs proved to be specific for the indicated target antigen. We conclude that the tested ABs are unsuitable for immunohistochemistry in paraffin-embedded murine tissues.

Identification of the Muscarinic Acetylcholine Receptor Subtype Mediating Cholinergic Vasodilation in Murine Retinal Arterioles

PURPOSE To identify the muscarinic acetylcholine receptor subtype that mediates cholinergic vasodilation in murine retinal arterioles. METHODS Muscarinic receptor gene expression was determined in murine retinal arterioles using real-time PCR. To assess the functional relevance of muscarinic receptors for mediating vascular responses, retinal vascular preparations from muscarinic receptor-deficient mice were studied in vitro. Changes in luminal arteriole diameter in response to muscarinic and nonmuscarinic vasoactive substances were measured by video microscopy. RESULTS Only mRNA for the M(3) receptor was detected in retinal arterioles. Thus, M(3) receptor-deficient mice (M3R(-/-)) and respective wild-type controls were used for functional studies. Acetylcholine concentration-dependently dilated retinal arterioles from wild-type mice. In contrast, vasodilation to acetylcholine was almost completely abolished in retinal arterioles from M3R(-/-) mice, whereas responses to the nitric oxide (NO) donor nitroprusside were retained. Carbachol, an acetylcholinesterase-resistant analog of acetylcholine, also evoked dilation in retinal arterioles from wild-type, but not from M3R(-/-), mice. Vasodilation responses from wild-type mice to acetylcholine were negligible after incubation with the non-subtype-selective muscarinic receptor blocker atropine or the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester, and were even reversed to contraction after endothelial damage with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. CONCLUSIONS These findings provide evidence that endothelial M(3) receptors mediate cholinergic vasodilation in murine retinal arterioles via activation of NO synthase.

Neuroprotection of medical IOP-lowering therapy

Intraocular pressure (IOP)-lowering therapy has been shown to arrest or retard the progression of optic neuropathy typical for glaucoma and can, thus, be described as neuroprotective. At present, six classes of medical therapy are employed, namely parasympathomimetics, alpha/beta-sympathomimetics, β-blockers, carbonic anhydrase inhibitors, α2-adrenergic receptor agonists and prostaglandin analogues. For several of these substances, some experimental evidence exists of a possible neuroprotective mechanism, beyond their IOP-lowering activity. β-Blockers are involved in the up-regulation of brain-derived neurotrophic factor (BDNF) and can decrease glutamate-mediated NMDA receptor activation. Not only systemic but also topical carbonic anhydrase inhibitors are able to increase retinal blood flow. α2-Adrenergic receptor agonists can up-regulate the formation of BDNF and anti-apoptotic factors. Prostaglandin analogues increase blood flow to the eye, possibly including the retina. To date, evidence for a neuroprotective effect independent of IOP regulation in human glaucoma is scarce and has only been shown to be likely for the α2-adrenergic receptor agonist, brimonidine.

Role of the M3 muscarinic acetylcholine receptor subtype in murine ophthalmic arteries after endothelial removal.

PURPOSE We tested the hypothesis that the M3 muscarinic acetylcholine receptor subtype mediates cholinergic responses in murine ophthalmic arteries after endothelial removal. METHODS Muscarinic receptor gene expression was determined in ophthalmic arteries with intact and with removed endothelium using real-time PCR. To examine the role of the M3 receptor in mediating vascular responses, ophthalmic arteries from M3 receptor-deficient mice (M3R(-/-)) and respective wild-type controls were studied in vitro. Functional studies were performed in nonpreconstricted arteries with either intact or removed endothelium using video microscopy. RESULTS In endothelium-intact ophthalmic arteries, mRNA for all five muscarinic receptor subtypes was detected, but M3 receptor mRNA was most abundant. In endothelium-removed ophthalmic arteries, M1, M2, and M3 receptors displayed similar mRNA expression levels, which were higher than those for M4 and M5 receptors. In functional studies, acetylcholine evoked vasoconstriction in endothelium-removed arteries from wild-type mice that was virtually abolished after incubation with the muscarinic receptor blocker atropine, indicative of the involvement of muscarinic receptors. In concentration-response experiments, acetylcholine and carbachol concentration-dependently constricted endothelium-removed ophthalmic arteries from wild-type mice, but produced only negligible responses in arteries from M3R(-/-) mice. In contrast, acetylcholine concentration-dependently dilated ophthalmic arteries with intact endothelium from wild-type mice, but not from M3R(-/-) mice. Responses to the nitric oxide donor nitroprusside and to KCl did not differ between ophthalmic arteries from wild-type and M3R(-/-) mice, neither in endothelium-intact nor in endothelium-removed arteries. CONCLUSIONS These findings provide evidence that in murine ophthalmic arteries the muscarinic M3 receptor subtype mediates cholinergic endothelium-dependent vasodilation and endothelium-independent vasoconstriction.

Intraocular pressure measurements during flap preparation using 2 femtosecond lasers and 1 microkeratome in human donor eyes

PURPOSE: To evaluate and compare intraocular pressures (IOPs) during flap preparations performed using 2 femtosecond lasers and a mechanical microkeratome in human donor globes. SETTING: University Medical Center Mainz, Mainz, and Euroeyes Clinic Stuttgart, Stuttgart, Germany. DESIGN: Experimental study. METHODS: A cannula was inserted through the optic nerve in human globes. The IOP was obtained continuously during flap preparation using the 60 kHz Intralase femtosecond laser, the 200 kHz Visumax femtosecond laser, or the Amadeus II microkeratome. For each experiment, a normal lamellar flap preparation (regular procedure) and a worst‐case procedure (femtosecond laser interface was pressed against globe until docking maneuver was aborted) were performed. RESULTS: During the regular procedure, the mean maximum IOP measured was 181.3 mm Hg (range 159.1 to 194.8 mm Hg) with the 60 kHz femtosecond laser, 77.6 mm Hg (range 58.1 to 100.3 mm Hg) with the 200 kHz femtosecond laser, and 198.1 mm Hg (range 162.8 to 299.6 mm Hg) with the microkeratome. During the worst‐case procedure, the maximum measured IOP was 319.7 mm Hg (range 299.1 to 341.2 mm Hg) with the 60 kHz laser and 120.4 mm Hg (range 118.1 to 134.7 mm Hg) with the 200 kHz laser. CONCLUSION: Maximum IOPs during corneal flap preparations in human enucleated eyes were lower during performance of a regular procedure and a worst‐case procedure with the 200 kHz femtosecond laser than with the 60 kHz femtosecond laser and the mechanical microkeratome. Financial Disclosure: Dr. Sekundo is a member of the Scientific Advisory Board of Carl Zeiss Meditec AG, Jena, Germany. No author has a financial or proprietary interest in any material or method mentioned.