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Biomedical subjects

A J Harmar

Publications and source records attributed to A J Harmar.

At least 19 recordsLinked to original sources
PubMed1998

Desensitization of the human vasoactive intestinal peptide receptor (hVIP2/PACAP R): evidence for agonist-induced receptor phosphorylation and internalization.

To investigate the role of phosphorylation and internalization in the desensitization of the hVIP2/PACAP receptor, we expressed a C-terminal epitope-tagged (hemagglutinin; YPYDVPDYASL) receptor in COS7 and HEK293 cell lines. Radiolabeling experiments demonstrated that exposure to agonist induced receptor phosphorylation significantly above basal levels. This receptor phosphorylation was greater than that induced by receptor-independent activation of PKA with forskolin and that induced by co-application of forskolin and agonist. This suggests that receptor occupancy promotes phosphorylation and also that receptor phosphorylation may involve a specific G protein-coupled receptor kinase in addition to PKA. Immunocytochemical analysis showed that the receptor was internalized in response to agonist to a single site of accumulation within the cell and this was dependent on temperature, agonist concentration, and time. Further studies will focus on identifying phosphorylation sites and endocytic signals within the hVIP2/PACAP R.

Animals↗
PubMed1998

Circadian changes in PACAP type 1 (PAC1) receptor mRNA in the rat suprachiasmatic and supraoptic nuclei.

A receptor for pituitary adenylate cyclase activating polypeptide (PACAP), denoted as PAC1, is expressed in the suprachiasmatic nuclei (SCN). Since the circadian clock demonstrates phase-dependent sensitivity to PACAP, we have used in situ hybridization histochemistry to examine whether PAC1 mRNA is differentially expressed in the rat SCN across the 24-h cycle. There was a significant variation in PAC1 mRNA within the SCN and supraoptic nuclei during the light-dark cycle and in constant darkness, with peaks at the middle of both the real and subjective day and night; no significant variation was observed in the cingulate cortex. The results suggest that the phase-dependent actions of PACAP on the clock may involve phase-specific changes in the availability of PAC1 receptors within the SCN.

Analysis of Variance↗
PubMed1998

Expression of PACAP, and PACAP type 1 (PAC1) receptor mRNA during development of the mouse embryo.

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have been reported to have a number of neurotrophic effects. We have examined the expression of mRNA for PACAP and PACAP type 1 (PAC1) receptor in the mouse embryo by in situ hybridization and the effects of PACAP and VIP on the growth of mouse embryos in vitro. Although we were unable to detect gross effects of either peptide on the growth rates of embryos maintained in culture, mRNAs for both PAC1 receptor and PACAP peptide were present in the nervous system from day 9.5 of embryonic development. PAC1 receptor mRNA was most abundant in the neural tube and the rhombencephalon and was present also in the dorsal root and trigeminal ganglia and the sympathetic chain. The distribution of mRNA for the PACAP peptide overlapped in part with that of the receptor, but was more extensively distributed in the rhombencephalon and in the developing hypothalamus. Within the neural tube, PAC1 receptor mRNA was located in the roof and floor plates, while the distribution of PACAP peptide mRNA was more complex, being located in two columns of cells in the ventromedial neural tube (consistent with the position of developing autonomic motor neurons) and in cells in the dorsolateral neural tube. These data are concordant with a role for PACAP or a related peptide in neural development.

Animals↗
PubMed1998

Circadian changes in the expression of vasoactive intestinal peptide 2 receptor mRNA in the rat suprachiasmatic nuclei.

The suprachiasmatic nuclei (SCN) in the hypothalamus function as the primary circadian pacemaker. A receptor for vasoactive intestinal peptide (VIP), denoted as VIP2, is abundantly expressed in the SCN. Since the rodent circadian clock demonstrates phase-dependent sensitivity to exogenous VIP, we investigated the possibility that VIP2 receptor mRNA is differentially expressed in the SCN across the 24 h cycle. To establish whether VIP2 receptor mRNA levels change across the 12:12 h light-dark (LD) cycle (lights on designated as Zeitgeber time (ZT)O), rats were killed at ZT 0, 2, 6, 10, 12, 14, 18 and 22. To determine if variation in this mRNA occurs in the absence of LD entrainment cues, lights were not turned on at the time of transition from dark to light (designated as CT O); the animals in this group were killed in constant darkness (DD) at CT 0, 2, 6, 10, 12, 14, 18 and 22. In situ hybridization histochemistry indicated no variations in VIP2 receptor mRNA in the cingulate cortex under either LD or DD conditions. There was, however, significant variation in the expression of VIP2 receptor mRNA within the SCN during the LD cycle, with one peak at ZT 6 and at ZT 22. A comparable biphasic pattern of mRNA expression was observed in DD animals with peaks at CT 10 and another at CT 22. The results suggest that the phase-dependent actions of VIP on the clock may involve phase-specific changes in the availability of VIP2 receptor within the SCN.

Animals↗
PubMed1998

Altered allelic distributions of the serotonin transporter gene in migraine without aura and migraine with aura.

Allelic variation of the human serotonin transporter gene (HSERT), a highly plausible candidate gene for susceptibility to migraine, was investigated in 266 individuals with migraine, including 173 having migraine without aura (MO), 94 having migraine with aura (MA), 18 with co-occurrence of MO and MA, plus 133 unaffected controls. The distribution of a polymorphism with different forms of a variable tandem number repeat (VNTR) in intron 2 were compared. The MO group had an over-representation of genotypes with two twelve repeat alleles (STin2.12) and a reduction of genotypes containing one ten repeat (STin2.10) compared to controls. The MA group showed a similar pattern, but also a trend towards an increase in genotypes containing the nine repeat allele of the VNTR (STin2.9). Genotypes containing this allele were found in 6.4% of the MA group compared to 2.3% of controls. The group with co-occurrence of MO and MA had a significantly different pattern of overall allele frequency distribution from controls, reflecting a reduction in genotypes containing the STin2.10 allele and a shift both to STin2.9 carriers and to STin2.12 homozygosity. These results support the view that susceptibility to MO and MA has a genetic component, that these disorders are distinct, and that genetic susceptibility may in some cases be associated with a locus at or near the serotonin transporter gene.

Adult↗
PubMed1997

Celsr1, a neural-specific gene encoding an unusual seven-pass transmembrane receptor, maps to mouse chromosome 15 and human chromosome 22qter.

We have identified Celsr1, a gene that encodes a developmentally regulated vertebrate seven-pass transmembrane protein. The extracellular domain of Celsr1 contains two regions each with homology to distinct classes of well-characterized motifs found in the extra-cellular domains of many cell surface molecules. The most N-terminal region contains a block of contiguous cadherin repeats, and C-terminal to this is a region containing seven epidermal growth factor-like repeats interrupted by two laminin A G-type repeats. Celsr1 is unique in that it contains this combination of repeats coupled to a seven-pass transmembrane domain. As part of the characterization of the Celsr1 gene, we have determined its chromosomal map location in both mouse and human. The European Collaborative Interspecific Backcross (EUCIB) and BXD recombinant inbred strains were used for mapping Celsr1 cDNA clones in the mouse, and fluorescence in situ hybridization was used to map human Celsr1 cosmid clones on metaphase chromosomes. We report that Celsr1 maps to proximal mouse Chromosome 15 and human chromosome 22qter, a region of conserved synteny. Reverse transcriptase-polymerase chain reaction analysis and in situ hybridization were used to determine the spatial restriction of Celsr1 transcripts in adult and embryonic mice. The results presented here extend our previous finding of expression of the Celsr1 receptor in the embryo and show that expression continues into adult life when expression in the brain is localized principally in the ependymal cell layer, choroid plexus, and the area postrema.

Animals↗
PubMed1996

Chromosomal localization in mouse and human of the vasoactive intestinal peptide receptor type 2 gene: a possible contributor to the holoprosencephaly 3 phenotype.

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) have been shown to act on a wide range of tissue and cell types, both in the central nervous system and in the periphery. Two distinct receptors for VIP, the VIP receptor type 1 (VIPR1) and the VIP receptor type 2 (VIPR2), have recently been cloned, each of which binds PACAP and VIP with equal affinity. We report here the chromosomal mapping of the human and mouse VIPR2 genes by fluorescence in situ hybridization. The VIPR2 gene maps to the human chromosomal region 7q36.3 and to the F2 region of mouse chromosome 12. Our localization of the human gene places it in the region where the locus for the craniofacial defect holoprosencephaly type 3 (HPE3) maps. Further mapping experiments, carried out on cell lines derived from patients with HPE or HPE microforms and associated 7q deletions, have led us to redefine the distal extent of the HPE3 minimal critical region, originally characterized by Gurrieri et al. (1993, Nature Genet. 3: 247-251.) The VIPR2 gene lies within this new HPE3 minimal critical region. Our results suggest that deletion of the VIPR2 gene is not the sole factor responsible for the HPE3 phenotype. However, it is possible that monosomy at the VIPR2 locus may contribute to the phenotype observed in many cases of HPE3.

Animals↗
PubMed1996

Expression of pituitary adenylate cyclase activating polypeptide receptors in the early mouse embryo as assessed by reverse transcription polymerase chain reaction and in situ hybridisation.

We have examined the expression of mRNA for the pituitary adenylate cyclase activating polypeptide (PACAP) receptor in the mouse from day 9.5 of embryonic development onwards by reverse transcription polymerase chain reaction (RT-PCR) and by in situ hybridisation. PACAP receptor mRNAs were detected by PCR in the earliest developmental stage examined and thereafter the levels of mRNA for all receptors increased during development. Wholemount in situ hybridisation first showed the expression of the PACAP receptor in the neural tube of 9.5 day old embryos, in later embryos heaviest expression of the PACAP receptor was found in the developing rhombencephalon (day 10.5 and 11.5). These results are consistent with a role for PACAP or a related peptide in the early development of the central nervous system.

Animals↗
PubMed1996

Polymorphism in serotonin transporter gene associated with susceptibility to major depression.

UNLABELLED: BACKGROUND; The serotonin transporter of the brain provides the primary target for the action of selective antidepressant drugs. We set out to identify polymorphisms of the serotonin transporter gene and to find out whether there was a relation between any such polymorphisms and the occurrence of affective disorder. METHODS: A comparison of a polymorphic region of the human serotonin transporter gene was carried out between two groups. The study group comprised 83 patients (39 unipolar depressive disorder, 44 bipolar disorder) with major affective disorder. The control group comprised 122 anonymous blood donors, and 71 volunteers who had been screened for psychiatric disorders. FINDINGS: We detected three novel alleles of the variable-number-tandem-repeat (VNTR) region (STin2.9, STin2.10) and Stin2.12) containing nine, ten and 12 copies of the VNTR element, respectively. The frequencies of the different forms of the allele in the control group were compared with those in the affective disorder group. There was a significant difference between the control and affective disorder groups, largely explained by the excess of the STin2.9 allele in the unipolar group (chi2=10.05, p<0.004 [Bonferroni corrected]). The presence of the allele with nine copies of the repeat was significantly associated with risk of unipolar disorder (odds ratio=6.95 [95% CI 1.8-27.2]). INTERPRETATION: This association, for an obvious candidate gene, may provide a critical starting point for an understanding of the likely polygenic contributions towards susceptibility to affective disorder.

Alleles↗
PubMed1996

Structure of a variable number tandem repeat of the serotonin transporter gene and association with affective disorder.

We have recently reported an association between a polymorphism of a variable number tandem repeat (VNTR) region of the serotonin transporter gene and susceptibility to major depressive disorder. We identified three alleles containing respectively 9 (STin2.9), 10 (STin2.10) and 12 (STin2.12) copies of a repetitive element. We report here the sequences of the three alleles. The repetitive element conformed to the consensus sequence, GGCTGYGACCY(R)GRRTG, where Y = T/C, R = G/A, with loss of the 12th base pair in one of the repeating elements. We have also extended the numbers of cases and controls in the study. The frequencies of the three alleles in 119 individuals with single or recurrent major depressive episodes, 128 individuals with bipolar disorder and a group of 346 controls were compared. There was a significant difference between patients with affective disorder and controls in the proportion of individuals carrying the STin2.9 allele. For the risk of unipolar disorder given a single STin2.9 allele, the odds ratio was 4.44 (95% Cl, 1.65-11.95) and for bipolar disorder 3.22 (95% Cl, 1.15-9.09). The findings support the hypothesis that allelic variation in the serotonin transporter gene may contribute to susceptibility for both major depression and bipolar disorder.

Alleles↗
PubMed1995

Control of a novel adenylyl cyclase by calcineurin.

The molecular complex formed by the immunosuppressant FK506 and the immunophilin protein FKBP12 potently inhibits the Ca2+/calmodulin-activated protein phosphatase calcineurin. This mechanism appears to be common to all types of cell, implying that fundamental physiological modes of calcineurin regulation are exploited by immunosuppressants. The present paper describes a novel adenylyl cyclase regulated by calcineurin that contains an FKBP12-like domain and may thus constitute a physiologically relevant calcineurin docking site mimicked by immunosuppressant-immunophilin complexes. The enzyme messenger RNA is particularly enriched in the cerebral cortex, striatum and hippocampus, where it is localized to neuronal perikarya, indicative of an important role in neuronal function.

Adenylyl Cyclases↗