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Biomedical subjects

M Wheatley

Publications and source records attributed to M Wheatley.

At least 73 records · Page 4Linked to original sources
PubMed1993

Fluorescent and biotinylated linear peptides as selective bifunctional ligands for the V1a vasopressin receptor.

We have designed and synthesized a linear peptide analogue of arginine vasopressin. This peptide, [1-phenylacetyl, 2-O-methyl-D-tyrosine, 6-arginine, 8-arginine, 9-lysinamide]vasopressin (PhAcALVP), has a lysinamide residue substituted for the more usual glycinamide at position 9. Derivatization of PhAcALVP at the N epsilon-lysyl amino group with N-hydroxysuccinimide esters of aminomethylcoumarin (Mec) and biotin (Btn) produced the bifunctional ligands PhAcAL(Mec)VP and PhAcAL(Btn)VP, respectively. Pharmacological characterization of these peptides revealed that all were high-affinity V1a-selective antagonists. PhAcAL(Btn)VP can simultaneously bind to both the rat liver V1a receptor and avidin conjugates. Using this strategy, we were able to study the distribution of V1a receptors on the surface of the rat mammary tumour cell line, WRK-1. Routine epifluorescent microscopy and confocal image analysis were used to observe the distribution of avidin-Texas-Red associated with receptor-bound PhAcAL(Btn)VP. We conclude that PhAcALVP is a useful precursor for the production of hetero-bifunctional V1a-selective ligands. Both PhAcAL(Mec)VP and PheAcAL(Btn)VP can be used selectively to probe the V1a receptor and will be versatile tools for a variety of histocytochemical applications, including receptor localization and purification.

Amino Acid Sequence↗
PubMed1993

Risk of blood contact through surgical gloves in aesthetic procedures.

Evidence of glove perforation, and therefore blood contact, was evaluated in 100 consecutive pairs of gloves used in aesthetic surgical procedures. The surgeon (38.3%) was more likely than the assistant (22.5%) to have exposure. In only 15% of these cases were they aware of the exposure. The left index finger (44%) was the most common location of perforation and 29 of the 32 holes (90.6%) were in cases that lasted more than 2 hours.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Hepatic vasopressin receptors (VPRs) exhibit species heterogeneity--absence of VPRs in sheep liver.

1. We have studied the binding characteristics of the hepatic VPRs expressed by rat, cow, pig, sheep and human and have demonstrated species heterogeneity. 2. These species differences are manifested as variation in the VPR capacity (rat > cow > pig > human > sheep), and in the affinity of these receptors for their natural ligand AVP (rat = human > pig = cow). 3. A single class of VPRs, with high affinity for AVP, is present in rat, cow, human and pig liver. In contrast, ovine hepatocytes do not express a VPR. 4. The affinity of the V1a-selective antagonist d(CH2)5Tyr(Me)2AVP is highly dependent on the species utilised, such that rat > human > cow.

Animals↗
PubMed1992

A comparison of intact and in-vitro locomotion in an adult amphibian.

Locomotion was compared in an intact and in-vitro preparation of the adult mudpuppy (Necturus maculatus). The intact animals walked on an aquatic treadmill while in-vitro preparations were made to walk with a bath application of the excitatory amino acid NMA (N-methyl DL-aspartate). EMG recordings of shoulder muscles (pectoralis, latissimus dorsi, dorsalis scapulae, and procoracohumeralis) and elbow muscles (brachialis and extensor ulnae) were obtained from intact animals while recordings were made from only the elbow muscles in-vitro. The in-vitro preparation required magnesium in the bath to initiate and maintain locomotion, consistent with an NMDA mediated response. Also consistent with an NMDA response was the finding that glycine potentiated the NMA induced locomotion in-vitro. The range of cycle durations seen in-vitro was well within the range seen in the intact animal, while gait analysis demonstrated the similarity of intact and in-vitro locomotor cycles. In spite of these very similar locomotor patterns there are interesting differences in the patterned output seen in-vitro, such as the absence of a second burst in the brachialis muscle in-vitro.

Animals↗
PubMed1992

An in vitro preparation of the mudpuppy for simultaneous intracellular and electromyographic recording during locomotion.

In this report we describe the development of an in vitro preparation of the mudpuppy (Necturus maculatus) used to investigate locomotion in walking vertebrates. The preparation consists of the first 5 segments of the cervical spinal cord and the attached forelimb. The preparation is bathed in a cooled (15 degrees C) and oxygenated spinal cord Ringers solution and remains viable for 36-100 h. Locomotion can be elicited during the first 36-48 h by applying the excitatory amino acid N-methyl D-aspartate (NMDA) to the bath. Cutaneous and dorsal root reflexes remain unchanged for much longer periods of time (72-100 h). During locomotion, intracellular recordings can be made from interneurons and motoneurons while simultaneous electromyographic (EMG) recordings are made from forelimb muscles. Rhythmically active interneurons can be classified according to their phase of activity during the step cycle. Further classification of interneurons involves both monitoring the afferent input to these cells from dorsal root and cutaneous afferents as well as using their action potentials as a trigger for averaging the ongoing locomotor EMG activity. In this way some of the input and output characteristics of the interneurons can be monitored. The ability to record simultaneously from interneurons and muscles offers distinct advantages over current in vitro preparations.

Animals↗
PubMed1992

Design and synthesis of heterofunctional V1a-selective vasopressin receptor ligands with lysine at position 9.

A peptide analogue of [8-arginine]vasopressin (AVP) with Lys substituted for Gly at position 9 ([d(CH2)5Tyr(Me)2LysNH2(9)]AVP; ALVP) has been synthesized as a precursor for the production of heterofunctional vasopressin receptor ligands. Three heterofunctional ligands have been prepared by attaching biotin and a photoreactive cross-linker capable of iodination, either alone or in combination, to the epsilon-amino group of Lys at position 9 in ALVP. The binding characteristics of these novel ligands have been determined at the V1a and V2 vasopressin receptors by employing membrane preparations of rat liver and kidney respectively. All of the analogues synthesized during the course of this study bound selectively, and with high affinity, to the V1a vasopressin receptor subtype. Our results demonstrate that the strategies described in this paper provide a convenient means of synthesizing heterofunctional vasopressin receptor ligands with preservation of subtype-specific, high affinity binding characteristics. These parameters establish the potential value of the analogues as probes for investigating V1a receptor structure and function.

Amino Acid Sequence↗
PubMed1991

Characterization of the human liver vasopressin receptor. Profound differences between human and rat vasopressin-receptor-mediated responses suggest only a minor role for vasopressin in regulating human hepatic function.

The [Arg8]vasopressin (AVP) receptor expressed by human hepatocytes was characterized, and compared with the rat hepatic V1a vasopressin receptor subtype. In addition to determining the pharmacological profile of the human receptor, the cellular responses to AVP were measured in human and rat hepatocytes by assaying glycogen phosphorylase alpha activity and DNA synthesis. Marked differences were observed between human and rat hepatocytes regarding vasopressin receptors and the intracellular consequences of stimulation by AVP. Data presented in this paper demonstrate the following, (i) Vasopressin V1a receptors are present in low abundance on human hepatocytes. (ii) Species differences exist between human and rat V1a receptors with respect to the affinity of some selective antagonists. (iii) AVP-stimulated glycogen phosphorylase a activation in human hepatocytes was approx. 5% of that observed in rat cells. (iv) In contrast with rat hepatocytes, DNA synthesis in human cells in culture was not stimulated by AVP. It is concluded that vasopressin plays only a minor role in the regulation of human hepatic function. Furthermore, conclusions drawn from observations made with AVP and its analogues on rat hepatic function cannot be directly extrapolated to the human situation.

Animals↗
PubMed1991

A selective biotinylated probe for V1a vasopressin receptors.

We have designed and synthesized a biotinylated vasopressin antagonist which is a selective probe for studying the V1a subtype of vasopressin receptor. Initially we synthesized the novel vasopressin analogue d(CH2)5Tyr(Me)2LysNH2(9)AVP (ALVP). Biotinamidocaproate was subsequently coupled to the epsilon-amino group of ALVP to generate the novel biotinylated probe d(CH2)5Tyr(Me)2Lys(N epsilon-biotinamido-caproate)NH2(9)AVP (ALBtnVP). Pharmacological characterization of ALVP and ALBtnVP established that both ligands were high affinity antagonists at V1a receptors, and that both displayed marked V1a/V2 selectivity. The observation that receptor-bound ALBtnVP was bi-functional, and thereby able to bind conjugated derivatives of avidin or streptavidin, allowed ALBtnVP to be utilized as a selective probe for V1a receptors. This strategy allowed the visualization of V1a receptors on the surface of WRK-1 cells and hippocampal neurons, by using streptavidin-gold with electron microscopy and fluorescein-avidin with light microscopy. We conclude that ALBtnVP is a useful probe for V1a receptors.

Amino Acid Sequence↗
PubMed1991

Growth of normal human hepatocytes in primary culture: effect of hormones and growth factors on DNA synthesis.

Although the ability of hormones and growth factors to stimulate DNA synthesis in rat hepatocytes has been investigated extensively, no such study of human hepatocytes has been reported. Here we describe a series of experiments to identify those factors that regulate human hepatocyte DNA synthesis in vitro and which therefore may play a role in the control of human liver regeneration. Human hepatocytes were isolated from normal liver tissue obtained after graft reduction for transplantation into pediatric recipients. Cells were maintained in culture for up to 5 days, and DNA synthesis was determined. Hydroxyurea reduced [3H]thymidine incorporation into DNA by 95%, indicating replicative DNA synthesis. As previously found with rat hepatocytes, epidermal growth factor and transforming growth factor-alpha stimulated DNA synthesis at low nanomolar concentrations; transforming growth factor-alpha was slightly more potent. Although the overall rate of thymidine incorporation was lower than that for rodent cells, human hepatocytes were sensitive to lower concentrations of these growth factors, and the degree of stimulation was similar. Conversely, transforming growth factor-beta inhibited DNA synthesis at low picomolar levels. By contrast (unlike rat hepatocytes), arginine-vasopressin failed to initiate or potentiate DNA synthesis in human cells. These data indicate that normal human hepatocytes can respond to low concentrations of growth promoters or inhibitors, previously shown to have activity on rat hepatocytes. These factors may then play a role in control of human liver growth. However, important species differences are apparent, highlighting the limitations of extrapolating from animal studies to humans.

Animals↗
PubMed1989

Propylbenzilylcholine mustard labels an acidic residue in transmembrane helix 3 of the muscarinic receptor.

Muscarinic acetylcholine receptors were purified from rat forebrain and labeled with [3H]N-(2-chloroethyl)N-(2',3'-[3H2]propyl)-2-aminoethylbenzilate. Cleavage of the labeled muscarinic acetylcholine receptors with a lysine-specific protease yielded labeled, glycosylated peptides about 130 and 200 residues in length, which came from different receptor sequences. The probable cleavage sites are in the second intracellular loop and in the second extracellular or third intracellular loop. The N-terminal 130 residues are disulfide-bonded to another part of the receptor structure, supporting the presence of a link between the second and third extracellular loops. The [3H]propylbenzilylcholine mustard-receptor link is cleaved by nucleophiles, acids, and bases under denaturing conditions, suggesting modification of an acidic residue. Cyanogen bromide cleavage points to transmembrane helix 3 as the site of label attachment.

Animals↗
PubMed1989

Binding and hydrodynamic properties of muscarinic receptor subtypes solubilized in 3-(3-cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate.

The muscarinic receptor from the cerebral cortex, heart, and lacrimal gland can be solubilized in the zwitterionic detergent 3-(3-cholamidopropyl)dimethylammonio-2-hydroxy-1-propane sulfonate (CHAPSO) with retention of high affinity [3H]N-methyls-copolamine binding. However, in this detergent there are significant differences in the binding properties of the receptors, compared with those observed in membranes and digitonin solution. Some agents retain a degree of selectivity. In the heart and cortex, agonists can bind with high affinity to a receptor-GTP-binding protein complex. A second, lower affinity, agonist binding state is also present, which resembles a class of sites seen in membranes but not in digitonin solution. The high affinity agonist binding state has been resolved from the lower affinity state on sucrose density gradient centrifugation. Hydrodynamic analysis suggests that the high affinity state is approximately 110,000 Da larger than the lower affinity state. The binding properties of the receptor in CHAPSO can be altered to those seen in digitonin by exchanging detergents after CHAPSO solubilization.

Animals↗
PubMed1989

Localization and structure of the muscarinic receptor ligand binding site.

A conserved aspartic acid residue in transmembrane helix 3 of the muscarinic acetylcholine receptors is important in binding the headgroup of muscarinic ligands. This acidic amino acid probably points into a relatively hydrophilic cavity whose walls are formed by the amphipathic transmembrane helices of the receptor. Amino acid side chains within this cavity contribute to ligand binding.

Binding Sites↗
PubMed1988

Muscarinic receptor subtypes and the selectivity of agonists and antagonists.

Receptors may be grouped into superfamilies, according to their mechanism. Muscarinic receptors belong to one such superfamily, that of the G-protein coupled receptors. Subtypes of muscarinic receptors may be defined according to their primary amino acid sequence, their mechanism and their pharmacology. However, there is as yet no consistent scheme to explain the pharmacology and function of each molecular subtype. The pharmacological tools which distinguish between muscarinic receptor subtypes are described and the emerging evidence for the location of the ligand binding site is discussed.

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗