Selective recognition of cyclic GMP using a fluorescence-based molecularly imprinted polymer

Guanosine 3′,5′-cyclic monophosphate (cGMP) plays a role as a second messenger in many different biological systems. Given the ubiquitous nature of cGMP, a simple method of detecting cGMP is of interest. To that end a fluorescent polymer with recognition sites for cGMP has been prepared. Its selectivity and sensitivity were investigated and a dose-dependant decrease in fluorescence of the polymer in the presence of cGMP was observed. In contrast, virtually no effect was detected upon application of the structurally similar molecules, guanosine 5′-monophosphate (GMP) and adenosine 3′,5′-cyclic monophosphate (cAMP), thus demonstrating the high selectivity of this polymer. The association constant for the binding of cGMP to the imprinted polymer was determined in order of 3 × 10 5 M -1. A fluorescent, molecularly imprinted polymer that selectively recognises cGMP may have a useful application as a fluorescent chemosensor for cGMP detection in biological samples.

An investigation into the synaptic conditions and celluar mechanisms underlying cerebellar synaptic plasticity

The direction of synaptic plasticity at the connection between parallel fibres (PFs) and Purkinje cells can be modified by PF stimulation alone. Strong activation (Hartell, 1996) or high frequency stimulation (Schreurs and Alkon, 1993) of PFs induced a long-term depression (LTD) of PF-mediated excitatory postsynaptic currents. Brief raised frequency molecular layer stimulation produced a cAMP-dependent long-temi potentiation (LTP) of field potential (FP) responses (Salin et al., 1998). Thin slices of cerebellar vermis were prepared from 14-21 day old male Wistar rats decapitated under Halothane anaesthesia. FP's were recorded from the Purkinje cell layer in response to alternate 0.2Hz activation of stimulating electrodes placed in the molecular layer. In the presence of picrotoxin, FPs displayed two tetrodotoxin-sensitive, negative-going components termed N1 and N2. EPs were graded responses with paired pulse facilitation and were selectively blocked by 101AM 6-cyano-7-nitroquinoxaline-2,3-dicne (CNQX) an antagonist at iy,-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type ionotropic glutamate receptors (AMPAR) suggesting that they were primarily PE-mediated. The effects of raised stimulus intensity (RS) and/or increased frequency (IF) activation of the molecular layer on FP responses were examined. In sagittai and transverse slices combined RS and IF molecular layer activation induced a LTD of the N2 component of FP responses. RSIF stimulation produced fewer incidences of LTD in sagittal slices when an inhibitor of nitric oxide synthase (NOS), guanylate cyclase (GC), protein kinase G (PKG) or the GABAB receptor antagonist CGP62349 was included into the perfusion medium. Application of a nitric oxide (NO) donor, a cyclic guanosine monophosphate (cGMP) analogue or a phosphodiesterase (PDE) type V inhibitor to prevent cGMP breakdown paired with IF stimulation produced an acute depression, Raised frequency (RF) molecular layer stimulation produced a slowly emerging LTD of N2 in sagittal slices that was largely blocked in the presence of NOS, cGMP or PKG inhibitors. In transverse slices RE stimulation produced a LTP of the N2 component that was prevented by an inhibitor of protein kinase A or NOS. Inhibition of cGMP-signalling frequently revealed an underlying potentiation suggesting that cGMP activity might mask the effects of cAMP. In sagittal slices RE stimulation resulted in a potentiation of FPs when the cAMP-specific PDE type IV inhibitor rolipram was incorporated into the perfusion medium. In summary, raised levels of PE stimulation can alter the synaptic efficacy at PF-Purkinje cell synapses. The results provide support for a role of NO/cGMP/PKG signalling in the induction of LTD in the cerebellar cortex and suggest that activation of GABAa receptors might also be important. The level of cyclic nucleotide-specific PDE activities may be crucial in determining the level of cGMP and CAMP activity and hence the direction of synaptic plasticity.

Pharmacological characterization of a receptor for calcitonin gene-related peptide on rat, L6 myocytes

1 The L6 myocyte cell line expresses high affinity receptors for calcitonin gene-related peptide (CGRP) which are coupled to activation of adenylyl cyclase. The biochemical pharmacology of these receptors has been examined by radioligand binding or adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation. 2 In intact cells at 37 degrees C, human and rat alpha- and beta-CGRP all activated adenylyl cyclase with EC50s of about 1.5 nM. A number of CGRP analogues containing up to five amino acid substitutions showed similar potencies. In membrane binding studies at 22 degrees C in 1 mM Mg2+, the above all bound to a single site with IC50s of 0.1-0.4 nM. 3 The fragment CGRP(8-37) acted as a competitive antagonist of CGRP stimulation of adenylyl cyclase with a calculated Kd of 5 nM. The Kd determined in membrane binding assays was lower (0.5 nM). 4 The N-terminal extended human alpha-CGRP analogue Tyro-CGRP activated adenylyl cyclase and inhibited [125I]-iodohistidyl-CGRP binding less potently than human alpha-CGRP (EC50 for cyclase = 12 nM, IC50 for binding = 4 nM). 5 The pharmacological profile of the L6 CGRP receptor suggests that it most closely resembles sites on skeletal muscle, cardiac myocytes and hepatocytes. The L6 cell line should be a stable homogeneous model system in which to study CGRP mechanisms and pharmacology."

Production and action of local mediators in the rat gastric mucosa

This study concerns the production and action of the local mediators nitric oxide (NO) and prostaglandin E2 (PGE2) in the rat gastric mucosa. The major objectives were: (i) to determine which mucosal cell type(s) contained NO synthase activity, (ii) to establish the functional role(s) of NO in the gastric mucosa and (iii) to investigate regulation of gastric PGE2 production. Gastric mucosal cells were isolated by pronase digestion coupled with intermittent calcium chelation and were separated by either density-gradient centrifugation or by counterflow elutriation. The distribution of Ca2+ -dependent NO synthase activity, measured via the conversion of [14C]-L-arginine to [14C]-L- citrulline, paralleled the distribution of mucous cells in elutriated fractions. Pre-treatment of rats with lipopolysaccharide caused the induction of Ca2+ -independent NO synthase in the elutriator fractions enriched with mucous cells. Incubation of isolated cells with the NO donor isosorbide dinitrate (ISDN) produced a concentration-dependent increase in the guanosine 3',-5'-cyclic monophosphate (cGMP) content which was accompanied by a concentration-dependent increase in release of immunoreactive mucin. Intragastric administration of ISDN of dibutyryl cGMP in vivo increased the thickness of the mucus layer overlying the gastric mucosa. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) produced a concentration-dependent inhibition (IC50 247 μM) of histamine-stimulated aminopyrine accumulation, a measure of secretory activity, in cell suspensions containing > 80% parietal cells. SNAP increased the cGMP content of the suspension but did not decrease cellular viability, glucose oxidation or adenosine 3',5'-cyclic monophosphate content. The inhibitory effect of SNAP was observed in permeabilised cells stimulated with ATP and was stereospecifically blocked by preincubation with Rp-8-bromoguanosine 3'-5'-monophosphorothioate, which inhibits activation of cGMP-dependent protein kinase. Stimulation of PGE2 release by bradykinin in a low density cell fraction, enriched with parietal cells and devoid of vascular endothelial cells and macrophages, involved a bradykinin B1 receptor. In summary, NO synthase activity is probably present in gastric mucous epithelial cells. NO may promote mucus secretion by elevation of cGMP. NO donors inhibit acid secretion at a specific site and their action may involve cGMP. The bradykinin B1 receptor is involved with PGE2 production in the gastric mucosa.