The M5 (m5) receptor subtype: fact or fiction?

By comparison to the other subtypes of muscarinic receptors, very little is known about the binding properties, locations, mechanisms and physiological functions of the M5 (m5)* receptor subtype. Studies of the m5 receptor have been hampered by the lack of m5-selective ligands or antibodies and a source that endogenously expresses predominantly the m5 receptor subtype. We have developed a pharmacological labeling strategy using the non-selective muscarinic antagonist [3H]NMS, in the presence of muscarinic antagonists and toxins in green mamba venom to occlude the m1-m4 receptor subtypes, to selectively label the m5 receptor subtype.

Glaucoma, capillaries and pericytes. 3. Peptide hormone binding and influence on pericytes.

To test the potential for vasoactive neuropeptide receptors to affect capillary resistance, we have begun to study the plausibility that pericytes might be equipped to respond to a representative peptide vasoconstrictor and a representative peptide vasodilator. Pericytes cultured from the bovine retinal vasculature specifically bind the angiotensin II (Ang II) antagonist saralasin (1 nM125I-saralasin bound at 2.2 +/- 0.

Differential regulation of molecular subtypes of muscarinic receptors in Alzheimer's disease.

Molecular subtypes of muscarinic receptors (m1-m5) are novel targets for cholinergic replacement therapies in Alzheimer's disease. However, the status of these receptors in human brain and Alzheimer's disease is incompletely understood. The m1-m5 receptors in brains from control subjects and Alzheimer's disease patients were examined using a panel of specific antisera and radioligand binding.

Differential alterations in muscarinic receptor subtypes in Alzheimer's disease: implications for cholinergic-based therapies.

Molecular subtypes of muscarinic receptors (m1-m5) are novel targets for cholinergic replacement therapies in Alzheimer's disease (AD). However, knowledge concerning the relative distribution, abundance and functional status of these receptors in human brain and AD is incomplete. Recent data from our laboratory have demonstrated a defect in the ability of the M1 receptor subtype to form a high affinity agonist-receptor-G protein complex in AD frontal cortex.

Attenuation of muscarinic receptor-G-protein interaction in Alzheimer disease.

Cortical M1 muscarinic receptor-G-protein coupling, high-affinity, guanine nucleotide-sensitive agonist binding (Flynn et al., 1991; Warpman et al., 1993) and muscarinic receptor-stimulated [3H]PIP2 hydrolysis (Ferrari-DiLeo and Flynn, 1993) are known to be defective in Alzheimer disease.

Selective labeling and localization of the M4 (m4) muscarinic receptor subtype.

We report here a novel strategy for the selective labeling and localization of the M4 (m4) muscarinic receptor subtype, based on the distinct kinetics of the muscarinic antagonists dexetimide and N-methylscopolamine (NMS) and on the selectivity profile of guanylpirenzepine and AF-DX 116 for the m1-m5 muscarinic receptor subtypes expressed in CHO-K1 cells. Incubation with 10 nM dexetimide, a nonselective antagonist, resulted in > 90% occupancy of each of the m1-m5 receptor subtypes. The relatively rapid rates of dexetimide dissociation from the m1, m2, and m4 receptor subtypes (t1/2 values of < 12.

Diminished muscarinic receptor-stimulated [3H]-PIP2 hydrolysis in Alzheimer's disease.

The functional integrity of the cortical muscarinic receptor (MR)-mediated phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C signalling pathway was assessed in Alzheimer disease (AD) and age-matched control subjects. There was no difference in the basal hydrolysis of [3H]-PIP2 to [3H]-inositol phosphates between control and AD membrane preparations. However, muscarinic agonist-stimulated PIP2 hydrolysis was significantly diminished in the AD cases.

Effects of cholinergic and adrenergic agonists on adenylate cyclase activity of retinal microvascular pericytes in culture.

Pericytes are contractile cells that might help regulate microvascular blood flow. To understand their potential role in the regulatory responses of the retina and optic nerve head vessels, the response of pericytes isolated from bovine retinal microvessels was determined to oxotremorine, isoproterenol, phenylephrine, and clonidine. Isoproterenol doubled the basal levels of cyclic adenosine monophosphate (cAMP) specifically through beta-adrenergic receptors, because the effect was blocked by dl-propranolol.

Beta-adrenergic responsiveness of choroidal vasculature.

Using in vitro binding methods and autoradiographs, the authors showed that choroidal vessels specifically bind iodine 125 cyanopindolol, a nonselective blocker of beta-adrenergic receptors, in albino rabbits. In humans, the presence of beta-adrenergic receptors in choroidal vessels was confirmed by showing an increased choroidal vascular tone after systemic administration of timolol maleate, a nonselective beta-adrenergic blocker. Topically administered timolol maleate lowered the intraocular pressure but did not reach the choroidal receptors in sufficient quantity to produce a measurable effect on vascular tone.

Angiotensin II binding receptors in retinal and optic nerve head blood vessels. An autoradiographic approach.

Angiotensin II (AII) binding sites were identified in cross-sections of the cat retinal and optic nerve vasculatures. The authors used 3H-AII and 125I-saralasin, an agonist and a high-affinity antagonist of AII receptors, respectively, to generate light microscopy autoradiograms in resin-embedded tissues. With both radioligands the presence of AII binding sites was confirmed in retinal arterioles but not in the veins or capillaries of the retina.

Response of retinal vasculature to phenylephrine.

In a freshly enucleated bovine eye, the retinal arteries and veins constrict when phenylephrine is applied directly on the surface of the vessel walls. This "in situ" preparation allowed us to observe the reaction of retinal vessels without any surgical procedure that might alter the vascular response to agonists. These results indicate that alpha1-adrenergic receptors are present in the retinal vessels, which might therefore respond to vasoactive hormones.

Biochemical evidence for cholinergic activity in retinal blood vessels.

Blood vessels from human, cat, pig and bovine retinas were analyzed for their contents of choline acetyltransferase (ChAT) and muscarinic binding sites. ChAT was measured by the synthesis of 3H-acetylcholine in the presence of 3H-acetyl CoA and choline. Muscarinic binding sites were determined by the specific binding of 3H-quinuclidinyl benzylate (3H-QNB).

Angiotensin-converting enzyme in bovine, feline, and human ocular tissues.

Angiotensin-converting enzyme was shown to be present in retinal vessels and neural retina of feline, bovine, and human eyes. It was also demonstrated in the other ocular tissues of feline eyes, in especially high concentration in the highly vascular uveal layer. Its role in the physiology of ocular blood flow and neurophysiology is uncertain, especially in the retina where circulating angiotensin and bradykinin are confined to the intravascular space by the blood-retina barrier, and sufficient data are not available to describe these peptides as transmitters or modulator molecules in the retina.

Beta 1 and beta 2 adrenergic binding sites in bovine retina and retinal blood vessels.

To supplement previous demonstration of angiotensin II and alpha-adrenergic binding sites, beta-adrenergic binding sites for dihydroalprenolol were demonstrated in vessel-free bovine neural retina (Kd = 2.5 nM) and bovine retinal vessels (Kd = 2.8 nM).

Angiotensin binding sites in bovine and human retinal blood vessels.

Blood vessels isolated from bovine and human retinas have sites that specifically bind 3H-angiotensin II (3H-Ag II) with an apparent dissociation constant (Kd) of 14 nM and a capacity of binding (Bmax) of 0.82 pmol/g. The binding sites for 3H-Ag II appear to be influenced by guanine nucleotides (GTP) and cations (Mg2+ and Na+) in a way that resembles angiotensin II receptors in other tissues.

Adrenergic alpha 1 and alpha 2 binding sites are present in bovine retinal blood vessels.

Bovine retinal vessels have sites that specifically bind 3H-p-aminoclonidine (3H-PAC) with an apparent dissociation constant of 0.12 nM and a capacity of binding of 0.15 pmol/g.

Alpha-adrenoreceptors and muscarine receptors in human pial arteries and microvessels: a receptor binding study.

Human pial arteries and intraparenchymal microvessels were isolated for enzyme assays and radioligand binding studies of receptors. Special attention was paid to contamination with brain tissue, which was assessed by luxol staining and cerebroside assays for myelin and by scanning electron microscopy. The amount of contamination was approximately 1% for pial vessels and 14% for microvessel preparations.