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M Wheatley

Publications and source records attributed to M Wheatley.

At least 55 records · Page 3Linked to original sources
PubMed1996

Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis.

Neural stem cells in the lateral ventricles of the adult mouse CNS participate in repopulation of forebrain structures in vivo and are amenable to in vitro expansion by epidermal growth factor (EGF). There have been no reports of stem cells in more caudal brain regions or in the spinal cord of adult mammals. In this study we found that although ineffective alone, EGF and basic fibroblast growth factor (bFGF) cooperated to induce the proliferation, self-renewal, and expansion of neural stem cells isolated from the adult mouse thoracic spinal cord. The proliferating stem cells, in both primary culture and secondary expanded clones, formed spheres of undifferentiated cells that were induced to differentiate into neurons, astrocytes, and oligodendrocytes. Neural stem cells, whose proliferation was dependent on EGF+bFGF, were also isolated from the lumbar/sacral segment of the spinal cord as well as the third and fourth ventricles (but not adjacent brain parenchyma). Although all of the stem cells examined were similarly multipotent and expandable, quantitative analyses demonstrated that the lateral ventricles (EGF-dependent) and lumbar/sacral spinal cord (EGF+bFGF-dependent) yielded the greatest number of these cells. Thus, the spinal cord and the entire ventricular neuroaxis of the adult mammalian CNS contain multipotent stem cells, present at variable frequency and with unique in vitro activation requirements.

Animals↗
PubMed1996

Molecular recognition of peptide and non-peptide ligands by the extracellular domains of neurohypophysial hormone receptors.

This study was designed to ascertain whether the extracellular loops of vasopressin/oxytocin receptors bind ligands and, if so, to locate the molecular determinants of this ligand-receptor interaction. Ligand-binding studies were employed using a rat liver V1a vasopressin receptor preparation and both peptide and non-peptide receptor ligands. Synthetic peptides corresponding to defined regions of the extracellular surface of the neurohypophysial hormone receptors recognized radioligands. These receptor mimetics inhibited the binding of radioligands to the V1a receptor with apparent affinities (pKi) ranging from 3.1 to 6.75. The same mimetics had no effects on the binding of angiotensin II to the rat AT1 receptor, indicating specificity for V1a receptor ligands. A mimetic peptide (DITYRFRGPDWL) of the first extracellular loop (ECII) of the V1a vasopressin receptor also inhibited vasopressin-stimulated, but not angiotensin II-stimulated, glycogen phosphorylase in isolated rat hepatocytes. In contrast, scrambled ECII mimetics displayed greatly reduced affinity for vasopressin. In addition, the role of peptide side-chain versus main-chain atoms in the binding of ligands by vasopressin receptors was addressed using retro-inverso peptide mimetics. Our findings indicate a precise orientation of the extracellular receptor surface (particularly the ECII domain) which facilitates the initial 'capture' of both peptide and non-peptide ligands. Moreover, the data indicate that the main-chain atoms of both a major binding-site determinant in the first extracellular loop of the receptor and the neurohypophysial hormones contribute significantly to the ligand-receptor interaction. These findings also suggest that soluble receptor-binding domains have therapeutic potential.

Amino Acid Sequence↗
PubMed1996

Probing the V1a vasopressin receptor binding site with pyroglutamate-substituted linear antagonists.

We have synthesized eight analogues of the linear vasopressin antagonist DTyr(Et)2-Phe3-Gln4-Asn5-Arg6-Pro7-Arg8-Tyr(NH2)9 substituted with L-, or D-, pyroglutamate at position-1, Asn or Val at position-4 and Arg or Met at position 6. All of these peptides bound to the V1a vasopressin receptor with affinities ranging 33.6-5, 470 nM. Of this series, only two peptides, [LpGlu1Val4Arg6Tyr(NH2)9]AVP Kd = 48.4 nM and [DpGlu1Val4Arg6Tyr(NH2)9]AVP Kd = 691 nM, bound to the V2 vasopressin receptor. All of the neurohypophysial hormone receptors studied (V1a VPR, V2 VPR and OTR) were found to be stereoselective with respect to the N-terminal pGlu residue. The effect on binding characteristics of L-pGlu1 and D-pGlu1 analogues was dependent on both the sequence of the peptide and on the receptor subtype in question. From these data we found that peptide 5, which has the structure DpGlu-DTyr(Et)-Phe-Val-Asn-Arg-Pro-ARg-Tyr(NH2), exhibited the highest V1a/OTR selectivity reported to date (V1aVPR Kd = 82 nM; OTR no binding at 10 microM). As such, peptide 5 will provide useful leads to the development of ligands with enhanced V1a/OTR selectivity. The binding affinity and hydrophobicity of pyroglutamate-substituted peptides was compared with previously characterized V1a receptor antagonists which contained a range of position-1 substitutions. The hydrophobicity of both cyclic and linear antagonists was markedly increased relative to the agonists AVP and [Phe2Orn8]VT but increased hydrophobicity alone did not exclusively lead to high affinity antagonists. Data presented support the contention that in addition to a general increase in hydrophobicity/lipophilicity, position-1 influences the pharmacophore of vasopressin antagonists by providing molecular determinants for ligand/receptor interaction.

Amino Acid Sequence↗
PubMed1996

Renal bradykinin and vasopressin receptors: ligand selectivity and classification.

We studied the specific binding of radiolabeled bradykinin ([3H]BK) and vasopressin ([3H]AVP) to membrane preparations of bovine and porcine kidney medulla. [3H]BK reversibly labeled a single site (Kd = 1.06 nM) in bovine kidney medulla independently of [Mg2+]. The number of BK receptors in bovine kidney medulla, Bmax = 122 fmol/mg protein, is markedly (2- to 3-fold) higher than that reported in other tissues. Further characterization by ligand binding indicated that the bovine bradykinin receptor was the B2a subtype, pharmacologically related to B2a receptors expressed by human and rabbit tissues. In contrast, the specific binding of [3H]BK, but not [3H]AVP, to porcine kidney medulla (Kd = 0.32 nM, Bmax = 45 fmol/mg) was dependent upon the presence of enzyme inhibitors to prevent the rapid and selective degradation of bradykinin. Interspecies differences were revealed for renal medulla V2 vasopressin receptors with respect to their abundance and their affinity for several V2-selective ligands. In summary, (i) bovine kidney medulla is a convenient source of tissue for studying the B2a bradykinin receptor subtype; (ii) there are significant species-dependent differences in both the abundance of renal medulla B2a and V2 receptors and the ligand selectivity of V2 receptors; and (iii) these findings are significant in relation to the physiological and pathological roles of renal kinins and their interaction with the neurohypophysial peptide hormone system.

Animals↗
PubMed1995

Molecular pharmacology of V1a vasopressin receptors.

1. Vasopressin, a mammalian neurohypophysial peptide hormone, has diverse physiological actions. 2. Pharmacological studies, using a range of mammalian tissues, have identified three subtypes of vasopressin receptor. 3. The V1a subtype of vasopressin receptor is widely distributed and mediates many central and peripheral actions of vasopressin. 4. The development of subtype-selective vasopressin analogues has provided valuable tools for pharmacological and physical studies of the V1a receptor protein. 5. Pharmacological differences indicate species heterogeneity in the characteristics of V1a receptors and in the expression of hepatic V1a receptors. 6. The cloning of neurohypophysial hormone receptor proteins allows structural and functional comparison of the V1a vasopressin receptors with other G-protein-coupled receptors.

Animals↗
PubMed1995

Intramedullary blood vessels of the spinal cord express V1a vasopressin receptors: visualization by a biotinylated ligand.

The neurohypophysial peptide hormone [Arg8]vasopressin (AVP) has well documented pressor effects in the periphery. These are mediated by vasopressin receptors (VPRs) of the V1a subtype, expressed by vascular smooth muscle cells, which induce vascular contraction when activated. AVP also has effects on the vasculature of the brain, where it has been reported to induce both vasodilation and vasoconstriction. The responsiveness of blood vessels of the spinal cord, however, has received little attention. To determine the morphology and distribution of blood vessels within the spinal cord, vessels were vizualised using a mouse anti-rat smooth muscle alpha actin IgG as primary antibody and fluorescein isothiocyanate-conjugated anti-mouse IgG secondary antibodies. A complementary vizualisation strategy which detected the endogenous peroxidase activity of red blood cells within vessels was also utilised. The characteristics of the structures observed using both visualisation strategies were typical of blood vessels. VPRs were localized using recently characterized high affinity biotinylated analogue of AVP (PhAcAL(Btn)VP), which is selective for the V1a subtype of VPR. PhAcAL(Btn)VP:VPR complexes were subsequently visualized by avidin-Texas red. The pharmacological characteristics of these sites were established using selective analogues of vasopressin and oxytocin. This confirmed that V1a receptors were indeed being visualized. The structures observed following visualization of VPRs had the same morphology as the vasculature revealed by the anti smooth muscle alpha-actin antibody. It can therefore be concluded that the blood vessels of the spinal cord express VPRs and are potentially responsive to AVP. Furthermore, VPRs were detected on capillaries of the microvasculature. As these capillaries are devoid of smooth muscle, VPRs must be expressed by endothelial cells as well as by smooth muscle cells. This distribution of VPRs would enable AVP to regulate local blood flow. The source of the AVP could be the general circulation, or perhaps more likely, to be local release from vasopressinergic hypothalamic neurones which are known to innervate specific regions of the spinal cord.

Animals↗
PubMed1995

Rat testicular myoid cells express vasopressin receptors: receptor structure, signal transduction, and developmental regulation.

Detection of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT) in the testis of several species has led to the proposal that these peptides may have a physiological role in the regulation of testicular function. Therefore, we investigated whether the contractile myoid cells of rat seminiferous tubules express functional receptors for AVP or OT and, thus, constitute a target for these hormones. This study used primary cultures of purified peritubular myoid cells derived from rats both before and after puberty. By several criteria, myoid cells prepared from adult rats expressed vasopressin receptors (VPRs). We detected specific and saturable [3H]AVP binding to a single population of sites with a Kd of 7.5 nM and a binding capacity of 145 fmol/mg protein. AVP stimulated the accumulation of inositol phosphates in a dose-dependent manner with an EC50 of 1.7 nM. Cloning and sequencing of the myoid cell VPR confirmed it to be the V1a subtype of VPR. VPR expression by myoid cells is under developmental control, as the receptors are present in the adult rat, but absent before puberty. In contrast, OT receptors were not expressed at any stage of development. Peritubular myoid cells are also responsive to endothelin-1 (ET-1), which potently stimulated phosphoinositidase-C. However, unlike AVP, the ET-1 responses were observed both before and after sexual maturity, suggesting different roles for AVP and ET-1 in the control of myoid cell function. Our data establish that the myoid cells of the adult rat seminiferous tubule are a target for AVP. This indicates an additional role for AVP in the regulation of testicular function and male fertility in the adult rat.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Pharmacological characterization of linear analogues of vasopressin generated by the systematic substitution of positions 1 and 6 by L-amino acids.

Eighteen linear analogues of [Arg8]vasopressin (AVP) were synthesized by systematically substituting the cysteine residues at positions 1 and 6 with a range of L-amino acids. Screening by competition ligand binding revealed that the combinations of amino acid residues tolerated at these positions was very restricted with respect to retention of vasopressin receptor (VPR) binding. Consequently, only three of the eighteen analogues investigated, [Pro1,Met6]AVP, [Gly1,Met6]AVP and [Phe1,Lys6]AVP, bound to the V1a receptor. Furthermore, these three peptides were all selective for the V1a receptor rather than the V1b, V2 and vasotocin receptors. In addition, although very homologous to the natural agonist, these analogues were in fact antagonists at V1a receptors. These data provide insights into the biophysical requirements at positions 1 and 6 of linear ligands for binding to V1a receptors and furthermore, supply clues to the nature of the receptor:ligand interaction.

Amino Acid Sequence↗
PubMed1994

Role of perivenous areolar tissue and recipient bed in the viability of venous flaps in the rabbit ear model.

This study was performed to investigate the role of perivenous areolar tissue and flap bed in the viability of venous flaps in the rabbit ear model as described by Inada et al. Six groups of flaps were studied: group A--flap based on a proximal vein and areolar tissue; group B--flap based on a proximal venous pedicle which has been skeletonised; group C--flaps based on a proximal vein and areolar tissue sutured over a full thickness skin graft (FTSG), preventing diffusion into the flap; group D--flaps with a skeletonised pedicle sutured over a FTSG; group E--control, non-vascularised flap placed directly on bed; and Group F--control, non-vascularised flap sutured over a FTSG. Groups C and D had 15 flaps and the remaining groups had 10 flaps in each. All flaps in groups A and B had total or partial survival. In group C 4 flaps survived completely, 8 had partial survival and 3 necrosed. Group D had only 2 flaps with partial survival with 13 flaps with total loss. In group E there were 10 flaps with partial survival and none with complete loss. No flaps in group F survived. Histological examination of the pedicle showed small vascular channels present in the areolar tissue surrounding the venous pedicle. This study confirms the importance of the perivenous areolar tissue in the perfusion of the venous skin flap in the rabbit ear model.

Animals↗
PubMed1994

The activity of interneurons during locomotion in the in vitro necturus spinal cord.

1. Less than two segments of the cervical spinal cord of the mudpuppy (Necturus maculatus) is sufficient to generate a locomotor rhythm with application of N-methyl-D-aspartic acid (NMDA). We have recorded intracellularly from rhythmically active interneurons in these segments and classified them according to their phase of firing within the step cycle and their afferent input. 2. Four classes of interneurons were found: flexor, flexor-->extensor, extensor, and extensor-->flexor. Interneurons that burst during the transition from flexion to extension or vice versa are referred to as "transitional" interneurons and represent the majority (68%) of rhythmically active interneurons studied in the mudpuppy spinal cord. 3. All flexor interneurons received only inhibitory input from cutaneous and dorsal root afferents, whereas the flexor-->extensor interneurons that responded received only excitatory input from dorsal root and cutaneous afferents. All extensor interneurons and all but one extensor-->flexor interneuron received no afferent input from the cutaneous or dorsal root afferents we stimulated. 4. Other interneurons have been classified as "tonic" cells. They fire continuously when the mudpuppy is walking and are silent when the mudpuppy is not walking. These interneurons receive no afferent input from the sources tested and may be responsible for turning locomotion on and off. 5. In conclusion, the presence of many transitional interneurons with specific patterns of afferent input may be required for the phasing of legged locomotion. We believe the in vitro preparation of the mudpuppy spinal cord and forelimb is an excellent model for studying the firing properties of interneurons during legged locomotion.

Afferent Pathways↗