The study aim was to determine if use of illicit amphetamines or ecstasy is associated with abnor... more The study aim was to determine if use of illicit amphetamines or ecstasy is associated with abnormal excitability of the corticomotoneuronal pathway and manipulation of novel objects with the hand. Methods Three groups of adults aged 18-50 years were investigated: individuals with a history of illicit amphetamine use, individuals with a history of ecstasy use but minimal use of other stimulants, and non-drug users. Transcranial magnetic stimulation was delivered to the motor cortex and the electromyographic response (motor evoked potential; MEP) was recorded from a contralateral hand muscle. Participants also gripped and lifted a novel experimental object consisting of two strain gauges and an accelerometer. Results Resting MEP amplitude was larger in the amphetamine group (6M, 6F) than the non-drug and ecstasy groups (p<0.005) in males but not females. Overestimation of grip force during manipulation of a novel object was observed in the amphetamine group (p=0.020) but not the ecstasy group. Conclusions History of illicit amphetamine use, in particular methamphetamine, is associated with abnormal motor cortical and/or corticomotoneuronal excitability in males and abnormal manipulation of novel objects in both males and females. Significance Abnormal excitability and hand function is evident months to years after cessation of illicit amphetamine use.
The sonographic appearance of the substantia nigra is abnormally bright and enlarged (hyperechoge... more The sonographic appearance of the substantia nigra is abnormally bright and enlarged (hyperechogenic) in young adults with a history of illicit stimulant use. The abnormality is a risk factor for Parkinson's disease. The aim of the current study was to identify the type of illicit stimulant drug associated with substantia nigra hyperechogenicity and to determine if individuals with a history of illicit stimulant use exhibit clinical signs of parkinsonism. We hypothesised that use of amphetamines (primarily methamphetamine) is associated with substantia nigra hyperechogenicity and clinical signs of parkinsonism. The area of echogenic signal in the substantia nigra was measured in abstinent human amphetamine users (n = 27; 33 ± 8 years) and in three control groups comprising a) 'ecstasy' users (n = 19; 23 ± 3 years), b) cannabis users (n = 30; 26 ± 8 years), and c) non-drug users (n = 37; 25 ± 7 years). A subset of subjects (n = 55) also underwent a neurological examinatio...
Journal of the Chemical Society, Chemical Communications, 1994
To explore the biological significance of the D-3 position phosphorylation of the second messenge... more To explore the biological significance of the D-3 position phosphorylation of the second messenger o-myo-inositol 1,4,5-trisphosphate (IP,) in cellular signalling, three novel D-3-substituted analogues of IP3 have been synthesized; their binding and Ca*+-release profiles at the IP3-receptor have been studied and shown to correlate with the steric requirement of the D-3 substituent.
Movement disorders : official journal of the Movement Disorder Society, 2011
The designation, DYT4, was assigned to an Australian family with whispering dysphonia. The role o... more The designation, DYT4, was assigned to an Australian family with whispering dysphonia. The role of known causes of dystonia has not been comprehensively investigated in this family, nor has the possible relationship with Wilson disease (WND) in 2 siblings. Eighteen family members were neurologically examined, and DNA samples were obtained. Linkage analysis was performed to DYT1, DYT6, DYT7, DYT11, DYT13, DYT15, and ATP7B with microsatellite markers and the THAP1 (DYT6), PRKRA (DYT16), and ATP7B (WND) genes were sequenced. Reevaluation of the family identified 9 living affected family members, 6 of whom are newly affected. Phenotypic expression was variable, ranging from isolated spasmodic dysphonia (often with mild craniocervical dystonia) to severe generalized dystonia. Two newly described features included an extrusional tongue dystonia and a unique "hobby horse gait." Genetic analyses excluded all tested loci. Haplotype analysis of the ATP7B region resulted in three dif...
Codeine is a popular opioid prodrug dependent on the activity of the specific cytochrome P450 enz... more Codeine is a popular opioid prodrug dependent on the activity of the specific cytochrome P450 enzyme 2D6 (CYP2D6). This enzyme catalyses the production of the potent analgesic metabolite morphine, but genetic studies have demonstrated that individuals from different ethnic groups exhibit considerable variability in the functional capacities of their expressed CYP2D6 enzymes, and pharmacological studies have shown many commonly prescribed drugs can reduce the action of CYP2D6 enzymes. These findings have significant clinical implications for the rational prescription of effective analgesia, especially in a multicultural country like Australia.
1. SchofieldMJ. HennrikusOJ.RedmanS, Sanson-Fisher RW. Prevalenceand characteristics of women who... more 1. SchofieldMJ. HennrikusOJ.RedmanS, Sanson-Fisher RW. Prevalenceand characteristics of women who have had a hysterectomy in a community survey. Aust N Z J Obstet Gynaeco/1991; 31: 153. 2. Carlson KJ. Outcomes of hysterectomy. Clin Obstet Gyneco/1997; 40: 939. 3. Reid BA, Aisbett CW, Jones LM, et al. Relative utilisation ratesof hysterectomy and linksto diagnosis.Canberra:Publication Production Unit, Commonwealth Department of Health and Aged Care, 2000. Q 3 (2%)
Methods in molecular biology (Clifton, N.J.), 1999
Many receptor tyrosine kinases and seven-transmembrane receptors are directly coupled or coupled ... more Many receptor tyrosine kinases and seven-transmembrane receptors are directly coupled or coupled via G proteins, respectively, to the activation of phosphoinositidase Cs. These enzymes catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the second messengers, myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P(3)] and diacylglycerol. Ins(1,4,5)P(3) interacts with a specific receptor that is a ligand-gated channel that allows mobilization of non-mitochondrial intracellular calcium (Ca(2+)) stores. Because Ins(1,4,5)P(3) is plasma membrane impermeant, this phenomenon was first demonstrated in permeabilized pancreatic acinar cells, and all subsequent studies in cells have involved introduction of Ins(1,4,5)P(3) by rendering a cell population permeable (3), using microinjection techniques, or by the presentation of chemically modified membrane-permeable Ins(1,4,5)P(3) analogs, such as photolabile &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;caged Ins(1,4,5)P(3)&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;. An alternative approach involves disruption of the plasma membrane and preparation of microsomes from the intracellular vesicular Ca(2+) stores; however, microsomal preparations exhibit a loss of Ins(1,4,5)P(3) responsiveness compared to permeabilized and intact cells.
Methods in molecular biology (Clifton, N.J.), 1999
Many cell surface receptors activate phosphoinositidase(s) C, via G proteins that catalyze the hy... more Many cell surface receptors activate phosphoinositidase(s) C, via G proteins that catalyze the hydrolysis of phosphatidylinositol 4,5-biphosphate to produce the second messengers, inositol(1,4,5)trisphosphate [Ins(1,4,5)P(3)] and diacylglycerol. Ins(1,4,5)P(3) interacts with specific receptor populations of ligand-gated channels to mobilize nonmitochondrial intracellular calcium (Ca(2+)) stores. Because Ins(1,4,5)P(3) is very hydrophilic, it cannot readily cross the intact plasma membrane. Consequently, Ins(1,4,5)P(3)-induced Ca(2+) release was initially demonstrated in permeabilized pancreatic acinar cells, and all subsequent studies in cells have involved the introduction of Ins(1,4,5)P(3) by rendering a cell population permeable, using microinjection techniques or by the presentation of chemically modified membrane-permeable Ins(1,4,5)P(3) analogs, such as photolabile &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;caged Ins(1,4,5)P(3)&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; (5). An alternative approach involves disruption of the plasma membrane and preparation of microsomes from the intracellular vesicular Ca(2+) stores, however, these preparations exhibit a loss of Ins(1,4,5)P(3) responsiveness compared to cells. The author will describe a (45)Ca(2+)-release assay used to monitor Ins(1,4,5)P(3)-induced Ca(2+) mobilization from nonmitochondrial intracellular Ca(2+) stores using &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;cytosol-like&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; buffer (CLB) and permeabilized SH-SY5Y neuroblastoma cell populations.
The interactions of synthetic L-chiro-inositol-2,3,5-trisphosphorothioate [L-ch-Ins(2,3,5)PS3] an... more The interactions of synthetic L-chiro-inositol-2,3,5-trisphosphorothioate [L-ch-Ins(2,3,5)PS3] and D-6-deoxy-myo-inositol-1,4,5-trisphosphorothioate [D-6-deoxy-Ins(1,4,5)PS3] with D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] receptors have been examined using radioligand binding assays and Ca2+ mobilization from permeabilized SH-SY5Y cells. The ability of these analogues to compete with [3H]Ins(1,4,5)P3 for specific sites on adrenal cortical membranes indicated that, although weaker than Ins(1,4,5)P3, both ligands competed fully for these sites [L-ch-Ins(2,3,5)PS3,Ki = 0.5 microM; D-6-deoxy-Ins(1,4,5)PS3,Ki = 5.3 microM]. However, in assays examining the amount of Ca2+ mobilized from the stores of permeabilized SH-SY5Y cells, both of these synthetic analogues displayed low intrinsic activity [L-ch-Ins(2,3,5)PS3, 34%; D-6-deoxy-Ins(1,4,5)PS3, 42% of that of Ins(1,4,5)P3]. Moreover, L-ch-Ins(2,3,5)PS3 and D-6-deoxy-Ins(1,4,5)PS3 were able to inhibit the response to Ins(1,4,5)P3 w...
Ins(1,3,4,5)P4 was able to mobilize the entire Ins(1,4,5)P3-sensitive intracellular Ca2+ store in... more Ins(1,3,4,5)P4 was able to mobilize the entire Ins(1,4,5)P3-sensitive intracellular Ca2+ store in saponin-permeabilized SH-SY5Y human neuroblastoma cells in a concentration-dependent manner, yielding an EC50 value of 2.05 +/- 0.45 microM, compared with 0.14 +/- 0.03 microM for Ins(1,4,5)P3. However, L-Ins(1,3,4,5)P4 [= D-Ins(1,3,5,6)P4] failed to cause mobilization of intracellular Ca2+ at concentrations up to 100 microM. Binding studies using pig cerebellar membranes as a source of both Ins(1,4,5)P3/Ins(1,3,4,5)P4-specific binding sites have revealed a marked contrast in their stereospecificity requirements. Ins(1,4,5)P3-receptors from pig cerebella exhibited stringent stereospecificity, L-Ins(1,4,5)P3 and L-Ins(1,3,4,5)P4 were > 1000-fold weaker, whereas Ins(1,3,4,5)P4 (IC50 762 +/- 15 nM) was only about 40-fold weaker than D-Ins(1,4,5)P3 (IC50 20.7 +/- 9.7 nM) at displacing specific [3H]Ins(1,4,5)P3 binding from an apparently homogeneous Ins(1,4,5)P3 receptor population. In co...
myo-Inositol-1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]-induced Ca2+ mobilization was examined in... more myo-Inositol-1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]-induced Ca2+ mobilization was examined in saponin-permeabilized SH-SY5Y cells using myo-inositol hexakisphosphate-supplemented buffer to prevent Ins(1,3,4,5)P4-3-phosphatase-catalyzed back-conversion of exogenous Ins(1,3,4,5)P4 to myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3]. The Ins(1,3,4,5)P4 concentration-response curve for Ca2+ release in SH-SY5Y cells exhibited an EC50 of 2.5 microM, compared with 52 nM for Ins(1,4,5)P3, with the maximally effective concentration of Ins(1,3,4,5)P4 (100 microM) mobilizing the entire Ins(1,4,5)P3-sensitive pool. Both Ins(1,3,4,5)P4- and Ins(1,4,5)P3-induced Ca2+ mobilizations were heparin sensitive. Further, L-chiro-inositol-2,3,5-trisphosphorothioate, a recently identified low intrinsic activity Ins(1,4,5)P3 receptor partial agonist, shifted both the Ins(1,4,5)P3 and Ins(1,3,4,5)P4 concentration-response curves significantly rightward, with similar potencies. However, binding studies dem...
Several novel D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3] analogues equatorially substitute... more Several novel D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3] analogues equatorially substituted at the 3-position have been synthesized to probe the structure-activity relationship of the Ins(1,4,5)P3-receptor subsite adjacent to the native 3-hydroxy (3-OH) of Ins(1,4,5)P3. This study was prompted, in part, by our observation that myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), the 3-position phosphorylated product of Ins(1,4,5)P3 was a full agonist at the Ca(2+)-mobilizing Ins(1,4,5)P3 receptor of SH-SY5Y cells (Wilcox, R.A., Challiss, R. A. J., Liu, C., Potter, B. V. I., and Nahorski, S. R. (1993) Mol. Pharmacol. 44, 810-817). The 3-position Ins(1,4,5)P3 analogues were equatorially substituted with groups spanning the steric range between the 3-OH of Ins(1,4,5)P3 and the 3-phosphate of Ins(1,3,4,5)P4; in order of increasing 3-position steric bulk these were: 3-fluoro-, 3-chloro-, 3-amino-, 3-bromo-, 3-methoxy-, and 3-phosphorothioate-Ins(1,4,5)P3. The analogues were as...
Journal of hypertension. Supplement : official journal of the International Society of Hypertension, 1994
VASCULAR SMOOTH MUSCLE AND THE PHOSPHOINOSITIDE SIGNALLING PATHWAY: Vascular smooth muscle tone c... more VASCULAR SMOOTH MUSCLE AND THE PHOSPHOINOSITIDE SIGNALLING PATHWAY: Vascular smooth muscle tone can be regulated by an array of agonists which act via receptor-mediated transmembrane signalling pathways to modify the concentrations of key intracellular second messengers. Following agonist stimulation the phosphoinositide signalling pathway initiates the contraction process in vascular smooth muscle, via the second messengers myo-inositol 1,4,5-trisphosphate and sn-1,2-diacylglycerol. DIVERSITY OF CALCIUM-REGULATORY MECHANISMS: The vascular smooth muscle cell apparently sustains the contraction with a number of diverse mechanisms, which act to increase intracellular Ca2+ by regulating both Ca2+ influx across the plasma membrane and Ca2+ release from intracellular calcium stores, or may act in the apparent absence of elevated cytosolic Ca2+ concentrations. The exact nature of these physiological interactions and their exact function are not yet fully understood. In particular, identif...
The novel, synthetic, adenophostin A analogue 2-hydroxyethyl-alpha-D-glucopyranoside-2,3',4&#... more The novel, synthetic, adenophostin A analogue 2-hydroxyethyl-alpha-D-glucopyranoside-2,3',4'-trisphosphate [Glu(2,3',4')P3] was synthesized to probe the structure-activity relationship at the D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R]. This study was stimulated by the recent observation that the fungal isolates adenophostins A and B were very potent, metabolically resistant, Ins(1,4,5)P3R agonists [J. Biol. Chem. 269:369-372 (1994)]. Gluc(2,3',4')P3 can be visualized as a truncated version of adenophostin A, in which the 2'- and 3'-carbons of the ribose ring, with their terminal phosphate groups, are retained and the remainder of the adenosine residue is excised. Gluc(2,3',4')P3 specifically displaced [3H]Ins(1,4,5)P3 from pig cerebellar Ins(1,4,5)P3 binding sites, with an affinity (IC50 = 130 nM) only 5-fold weaker than that of Ins(1,4,5)P3 (IC50 = 27 nM). Gluc(2,3',4')P3 was also a full agonist for Ca2...
The study aim was to determine if use of illicit amphetamines or ecstasy is associated with abnor... more The study aim was to determine if use of illicit amphetamines or ecstasy is associated with abnormal excitability of the corticomotoneuronal pathway and manipulation of novel objects with the hand. Methods Three groups of adults aged 18-50 years were investigated: individuals with a history of illicit amphetamine use, individuals with a history of ecstasy use but minimal use of other stimulants, and non-drug users. Transcranial magnetic stimulation was delivered to the motor cortex and the electromyographic response (motor evoked potential; MEP) was recorded from a contralateral hand muscle. Participants also gripped and lifted a novel experimental object consisting of two strain gauges and an accelerometer. Results Resting MEP amplitude was larger in the amphetamine group (6M, 6F) than the non-drug and ecstasy groups (p<0.005) in males but not females. Overestimation of grip force during manipulation of a novel object was observed in the amphetamine group (p=0.020) but not the ecstasy group. Conclusions History of illicit amphetamine use, in particular methamphetamine, is associated with abnormal motor cortical and/or corticomotoneuronal excitability in males and abnormal manipulation of novel objects in both males and females. Significance Abnormal excitability and hand function is evident months to years after cessation of illicit amphetamine use.
The sonographic appearance of the substantia nigra is abnormally bright and enlarged (hyperechoge... more The sonographic appearance of the substantia nigra is abnormally bright and enlarged (hyperechogenic) in young adults with a history of illicit stimulant use. The abnormality is a risk factor for Parkinson's disease. The aim of the current study was to identify the type of illicit stimulant drug associated with substantia nigra hyperechogenicity and to determine if individuals with a history of illicit stimulant use exhibit clinical signs of parkinsonism. We hypothesised that use of amphetamines (primarily methamphetamine) is associated with substantia nigra hyperechogenicity and clinical signs of parkinsonism. The area of echogenic signal in the substantia nigra was measured in abstinent human amphetamine users (n = 27; 33 ± 8 years) and in three control groups comprising a) 'ecstasy' users (n = 19; 23 ± 3 years), b) cannabis users (n = 30; 26 ± 8 years), and c) non-drug users (n = 37; 25 ± 7 years). A subset of subjects (n = 55) also underwent a neurological examinatio...
Journal of the Chemical Society, Chemical Communications, 1994
To explore the biological significance of the D-3 position phosphorylation of the second messenge... more To explore the biological significance of the D-3 position phosphorylation of the second messenger o-myo-inositol 1,4,5-trisphosphate (IP,) in cellular signalling, three novel D-3-substituted analogues of IP3 have been synthesized; their binding and Ca*+-release profiles at the IP3-receptor have been studied and shown to correlate with the steric requirement of the D-3 substituent.
Movement disorders : official journal of the Movement Disorder Society, 2011
The designation, DYT4, was assigned to an Australian family with whispering dysphonia. The role o... more The designation, DYT4, was assigned to an Australian family with whispering dysphonia. The role of known causes of dystonia has not been comprehensively investigated in this family, nor has the possible relationship with Wilson disease (WND) in 2 siblings. Eighteen family members were neurologically examined, and DNA samples were obtained. Linkage analysis was performed to DYT1, DYT6, DYT7, DYT11, DYT13, DYT15, and ATP7B with microsatellite markers and the THAP1 (DYT6), PRKRA (DYT16), and ATP7B (WND) genes were sequenced. Reevaluation of the family identified 9 living affected family members, 6 of whom are newly affected. Phenotypic expression was variable, ranging from isolated spasmodic dysphonia (often with mild craniocervical dystonia) to severe generalized dystonia. Two newly described features included an extrusional tongue dystonia and a unique "hobby horse gait." Genetic analyses excluded all tested loci. Haplotype analysis of the ATP7B region resulted in three dif...
Codeine is a popular opioid prodrug dependent on the activity of the specific cytochrome P450 enz... more Codeine is a popular opioid prodrug dependent on the activity of the specific cytochrome P450 enzyme 2D6 (CYP2D6). This enzyme catalyses the production of the potent analgesic metabolite morphine, but genetic studies have demonstrated that individuals from different ethnic groups exhibit considerable variability in the functional capacities of their expressed CYP2D6 enzymes, and pharmacological studies have shown many commonly prescribed drugs can reduce the action of CYP2D6 enzymes. These findings have significant clinical implications for the rational prescription of effective analgesia, especially in a multicultural country like Australia.
1. SchofieldMJ. HennrikusOJ.RedmanS, Sanson-Fisher RW. Prevalenceand characteristics of women who... more 1. SchofieldMJ. HennrikusOJ.RedmanS, Sanson-Fisher RW. Prevalenceand characteristics of women who have had a hysterectomy in a community survey. Aust N Z J Obstet Gynaeco/1991; 31: 153. 2. Carlson KJ. Outcomes of hysterectomy. Clin Obstet Gyneco/1997; 40: 939. 3. Reid BA, Aisbett CW, Jones LM, et al. Relative utilisation ratesof hysterectomy and linksto diagnosis.Canberra:Publication Production Unit, Commonwealth Department of Health and Aged Care, 2000. Q 3 (2%)
Methods in molecular biology (Clifton, N.J.), 1999
Many receptor tyrosine kinases and seven-transmembrane receptors are directly coupled or coupled ... more Many receptor tyrosine kinases and seven-transmembrane receptors are directly coupled or coupled via G proteins, respectively, to the activation of phosphoinositidase Cs. These enzymes catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the second messengers, myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P(3)] and diacylglycerol. Ins(1,4,5)P(3) interacts with a specific receptor that is a ligand-gated channel that allows mobilization of non-mitochondrial intracellular calcium (Ca(2+)) stores. Because Ins(1,4,5)P(3) is plasma membrane impermeant, this phenomenon was first demonstrated in permeabilized pancreatic acinar cells, and all subsequent studies in cells have involved introduction of Ins(1,4,5)P(3) by rendering a cell population permeable (3), using microinjection techniques, or by the presentation of chemically modified membrane-permeable Ins(1,4,5)P(3) analogs, such as photolabile &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;caged Ins(1,4,5)P(3)&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;. An alternative approach involves disruption of the plasma membrane and preparation of microsomes from the intracellular vesicular Ca(2+) stores; however, microsomal preparations exhibit a loss of Ins(1,4,5)P(3) responsiveness compared to permeabilized and intact cells.
Methods in molecular biology (Clifton, N.J.), 1999
Many cell surface receptors activate phosphoinositidase(s) C, via G proteins that catalyze the hy... more Many cell surface receptors activate phosphoinositidase(s) C, via G proteins that catalyze the hydrolysis of phosphatidylinositol 4,5-biphosphate to produce the second messengers, inositol(1,4,5)trisphosphate [Ins(1,4,5)P(3)] and diacylglycerol. Ins(1,4,5)P(3) interacts with specific receptor populations of ligand-gated channels to mobilize nonmitochondrial intracellular calcium (Ca(2+)) stores. Because Ins(1,4,5)P(3) is very hydrophilic, it cannot readily cross the intact plasma membrane. Consequently, Ins(1,4,5)P(3)-induced Ca(2+) release was initially demonstrated in permeabilized pancreatic acinar cells, and all subsequent studies in cells have involved the introduction of Ins(1,4,5)P(3) by rendering a cell population permeable, using microinjection techniques or by the presentation of chemically modified membrane-permeable Ins(1,4,5)P(3) analogs, such as photolabile &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;caged Ins(1,4,5)P(3)&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; (5). An alternative approach involves disruption of the plasma membrane and preparation of microsomes from the intracellular vesicular Ca(2+) stores, however, these preparations exhibit a loss of Ins(1,4,5)P(3) responsiveness compared to cells. The author will describe a (45)Ca(2+)-release assay used to monitor Ins(1,4,5)P(3)-induced Ca(2+) mobilization from nonmitochondrial intracellular Ca(2+) stores using &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;cytosol-like&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; buffer (CLB) and permeabilized SH-SY5Y neuroblastoma cell populations.
The interactions of synthetic L-chiro-inositol-2,3,5-trisphosphorothioate [L-ch-Ins(2,3,5)PS3] an... more The interactions of synthetic L-chiro-inositol-2,3,5-trisphosphorothioate [L-ch-Ins(2,3,5)PS3] and D-6-deoxy-myo-inositol-1,4,5-trisphosphorothioate [D-6-deoxy-Ins(1,4,5)PS3] with D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] receptors have been examined using radioligand binding assays and Ca2+ mobilization from permeabilized SH-SY5Y cells. The ability of these analogues to compete with [3H]Ins(1,4,5)P3 for specific sites on adrenal cortical membranes indicated that, although weaker than Ins(1,4,5)P3, both ligands competed fully for these sites [L-ch-Ins(2,3,5)PS3,Ki = 0.5 microM; D-6-deoxy-Ins(1,4,5)PS3,Ki = 5.3 microM]. However, in assays examining the amount of Ca2+ mobilized from the stores of permeabilized SH-SY5Y cells, both of these synthetic analogues displayed low intrinsic activity [L-ch-Ins(2,3,5)PS3, 34%; D-6-deoxy-Ins(1,4,5)PS3, 42% of that of Ins(1,4,5)P3]. Moreover, L-ch-Ins(2,3,5)PS3 and D-6-deoxy-Ins(1,4,5)PS3 were able to inhibit the response to Ins(1,4,5)P3 w...
Ins(1,3,4,5)P4 was able to mobilize the entire Ins(1,4,5)P3-sensitive intracellular Ca2+ store in... more Ins(1,3,4,5)P4 was able to mobilize the entire Ins(1,4,5)P3-sensitive intracellular Ca2+ store in saponin-permeabilized SH-SY5Y human neuroblastoma cells in a concentration-dependent manner, yielding an EC50 value of 2.05 +/- 0.45 microM, compared with 0.14 +/- 0.03 microM for Ins(1,4,5)P3. However, L-Ins(1,3,4,5)P4 [= D-Ins(1,3,5,6)P4] failed to cause mobilization of intracellular Ca2+ at concentrations up to 100 microM. Binding studies using pig cerebellar membranes as a source of both Ins(1,4,5)P3/Ins(1,3,4,5)P4-specific binding sites have revealed a marked contrast in their stereospecificity requirements. Ins(1,4,5)P3-receptors from pig cerebella exhibited stringent stereospecificity, L-Ins(1,4,5)P3 and L-Ins(1,3,4,5)P4 were > 1000-fold weaker, whereas Ins(1,3,4,5)P4 (IC50 762 +/- 15 nM) was only about 40-fold weaker than D-Ins(1,4,5)P3 (IC50 20.7 +/- 9.7 nM) at displacing specific [3H]Ins(1,4,5)P3 binding from an apparently homogeneous Ins(1,4,5)P3 receptor population. In co...
myo-Inositol-1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]-induced Ca2+ mobilization was examined in... more myo-Inositol-1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]-induced Ca2+ mobilization was examined in saponin-permeabilized SH-SY5Y cells using myo-inositol hexakisphosphate-supplemented buffer to prevent Ins(1,3,4,5)P4-3-phosphatase-catalyzed back-conversion of exogenous Ins(1,3,4,5)P4 to myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3]. The Ins(1,3,4,5)P4 concentration-response curve for Ca2+ release in SH-SY5Y cells exhibited an EC50 of 2.5 microM, compared with 52 nM for Ins(1,4,5)P3, with the maximally effective concentration of Ins(1,3,4,5)P4 (100 microM) mobilizing the entire Ins(1,4,5)P3-sensitive pool. Both Ins(1,3,4,5)P4- and Ins(1,4,5)P3-induced Ca2+ mobilizations were heparin sensitive. Further, L-chiro-inositol-2,3,5-trisphosphorothioate, a recently identified low intrinsic activity Ins(1,4,5)P3 receptor partial agonist, shifted both the Ins(1,4,5)P3 and Ins(1,3,4,5)P4 concentration-response curves significantly rightward, with similar potencies. However, binding studies dem...
Several novel D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3] analogues equatorially substitute... more Several novel D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3] analogues equatorially substituted at the 3-position have been synthesized to probe the structure-activity relationship of the Ins(1,4,5)P3-receptor subsite adjacent to the native 3-hydroxy (3-OH) of Ins(1,4,5)P3. This study was prompted, in part, by our observation that myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), the 3-position phosphorylated product of Ins(1,4,5)P3 was a full agonist at the Ca(2+)-mobilizing Ins(1,4,5)P3 receptor of SH-SY5Y cells (Wilcox, R.A., Challiss, R. A. J., Liu, C., Potter, B. V. I., and Nahorski, S. R. (1993) Mol. Pharmacol. 44, 810-817). The 3-position Ins(1,4,5)P3 analogues were equatorially substituted with groups spanning the steric range between the 3-OH of Ins(1,4,5)P3 and the 3-phosphate of Ins(1,3,4,5)P4; in order of increasing 3-position steric bulk these were: 3-fluoro-, 3-chloro-, 3-amino-, 3-bromo-, 3-methoxy-, and 3-phosphorothioate-Ins(1,4,5)P3. The analogues were as...
Journal of hypertension. Supplement : official journal of the International Society of Hypertension, 1994
VASCULAR SMOOTH MUSCLE AND THE PHOSPHOINOSITIDE SIGNALLING PATHWAY: Vascular smooth muscle tone c... more VASCULAR SMOOTH MUSCLE AND THE PHOSPHOINOSITIDE SIGNALLING PATHWAY: Vascular smooth muscle tone can be regulated by an array of agonists which act via receptor-mediated transmembrane signalling pathways to modify the concentrations of key intracellular second messengers. Following agonist stimulation the phosphoinositide signalling pathway initiates the contraction process in vascular smooth muscle, via the second messengers myo-inositol 1,4,5-trisphosphate and sn-1,2-diacylglycerol. DIVERSITY OF CALCIUM-REGULATORY MECHANISMS: The vascular smooth muscle cell apparently sustains the contraction with a number of diverse mechanisms, which act to increase intracellular Ca2+ by regulating both Ca2+ influx across the plasma membrane and Ca2+ release from intracellular calcium stores, or may act in the apparent absence of elevated cytosolic Ca2+ concentrations. The exact nature of these physiological interactions and their exact function are not yet fully understood. In particular, identif...
The novel, synthetic, adenophostin A analogue 2-hydroxyethyl-alpha-D-glucopyranoside-2,3',4&#... more The novel, synthetic, adenophostin A analogue 2-hydroxyethyl-alpha-D-glucopyranoside-2,3',4'-trisphosphate [Glu(2,3',4')P3] was synthesized to probe the structure-activity relationship at the D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R]. This study was stimulated by the recent observation that the fungal isolates adenophostins A and B were very potent, metabolically resistant, Ins(1,4,5)P3R agonists [J. Biol. Chem. 269:369-372 (1994)]. Gluc(2,3',4')P3 can be visualized as a truncated version of adenophostin A, in which the 2'- and 3'-carbons of the ribose ring, with their terminal phosphate groups, are retained and the remainder of the adenosine residue is excised. Gluc(2,3',4')P3 specifically displaced [3H]Ins(1,4,5)P3 from pig cerebellar Ins(1,4,5)P3 binding sites, with an affinity (IC50 = 130 nM) only 5-fold weaker than that of Ins(1,4,5)P3 (IC50 = 27 nM). Gluc(2,3',4')P3 was also a full agonist for Ca2...
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Papers by Robert Wilcox