-
Circulation Research May 2010Transient receptor potential melastatin (TRPM)3 is a calcium-permeable ion channel activated by the neurosteroid pregnenolone sulfate and positively coupled to insulin...
RATIONALE
Transient receptor potential melastatin (TRPM)3 is a calcium-permeable ion channel activated by the neurosteroid pregnenolone sulfate and positively coupled to insulin secretion in beta cells. Although vascular TRPM3 mRNA has been reported, there is no knowledge of TRPM3 protein or its regulation and function in the cardiovascular system.
OBJECTIVE
To determine the relevance and regulation of TRPM3 in vascular biology.
METHODS AND RESULTS
TRPM3 expression was detected at mRNA and protein levels in contractile and proliferating vascular smooth muscle cells. Calcium entry evoked by pregnenolone sulfate or sphingosine was suppressed by TRPM3 blocking antibody or knock-down of TRPM3 by RNA interference. Low-level constitutive TRPM3 activity was also detected. In proliferating cells, channel activity was coupled negatively to interleukin-6 secretion via a calcium-dependent mechanism. In freshly isolated aorta, TRPM3 positively modulated contractile responses independently of L-type calcium channels. Concentrations of pregnenolone sulfate required to evoke responses were higher than the known plasma concentrations of the steroids, leading to a screen for other stimulators. beta-Cyclodextrin was one of few stimulators of TRPM3, revealing the channels to be partially suppressed by endogenous cholesterol, the precursor of pregnenolone. Elevation of cholesterol further suppressed channel activity and loading with cholesterol to generate foam cells precluded observation of TRPM3 activity.
CONCLUSIONS
The data suggest functional relevance of TRPM3 in contractile and proliferating phenotypes of vascular smooth muscle cells, significance of constitutive channel activity, regulation by cholesterol, and potential value of pregnenolone sulfate in therapeutic vascular modulation.
Topics: Animals; Cell Line; Cell Proliferation; Cholesterol; Humans; Mice; Muscle Contraction; Muscle, Smooth, Vascular; Pregnenolone; RNA, Messenger; Structure-Activity Relationship; TRPM Cation Channels
PubMed: 20360246
DOI: 10.1161/CIRCRESAHA.110.219329 -
Psychopharmacology Jan 2023Chronic alcohol intake down-regulates GABAergic transmission and reduces levels of neuroactive steroids. These changes are associated with greater stress dysregulation... (Randomized Controlled Trial)
Randomized Controlled Trial
RATIONALE
Chronic alcohol intake down-regulates GABAergic transmission and reduces levels of neuroactive steroids. These changes are associated with greater stress dysregulation and high alcohol craving which in turn increases relapse risk.
OBJECTIVES
This study tested whether potentiation of the neurosteroid system with pregnenolone (PREG), a precursor to neuroactive steroids and known to increase GABAergic transmission, will normalize chronic alcohol-related stress adaptations in the hypothalamic-pituitary-adrenal (HPA) axis and autonomic responses and reduce alcohol craving to significantly impact relapse risk.
METHODS
Forty-three treatment-seeking individuals with alcohol use disorder (AUD) were randomized to placebo (PBO) or supraphysiologic pregnenolone doses of 300 mg or 500 mg treatment using a parallel-between subject design as part of a larger 8-week pilot clinical trial. In week 2, they participated in a 3-day laboratory experiment where on each day they self-administered the assigned study drug in the laboratory and were then exposed to 5-min personalized guided imagery provocation of stress, alcohol, or neutral/relaxing cues, one condition per day on separate days, in a random, counterbalanced order. Repeated assessments of alcohol craving, anxiety, HPA axis, heart rate (HR), systolic (SBP), and diastolic blood pressure (DBP) and serum pregnenolone levels were made on each day.
RESULTS
Pregnenolone levels were significantly increased in the PREG groups versus PBO. PREG treatment decreased stress- and alcohol cue- induced craving and dose-specifically reduced stress-induced anxiety in the 300 mg/day group. Both PREG doses compared to PBO also normalized CORT/ACTH and increased stress-induced HR, stress- and cue-induced SBP, and in the 300 mg PREG group cue-induced DBP responses relative to neutral condition.
CONCLUSIONS
Findings indicate that pregnenolone decreases stress- and alcohol cue-provoked craving and normalizes HPA axis and autonomic arousal in individuals with AUD, thereby supporting the need for further assessment of pregnenolone in the treatment of AUD.
Topics: Humans; Alcoholism; Craving; Hypothalamo-Hypophyseal System; Pregnenolone; Neurosteroids; Pituitary-Adrenal System; Anxiety; Alcohol Drinking; Ethanol; Arousal; Recurrence; Cues
PubMed: 36445398
DOI: 10.1007/s00213-022-06278-3 -
Molecular and Cellular Endocrinology Feb 2017Cytochrome P450 17A1 (CYP17A1) operates at the core of human steroidogenesis, directing precursors into mineralocorticoids, glucocorticoids, or sex steroids. Although... (Review)
Review
Cytochrome P450 17A1 (CYP17A1) operates at the core of human steroidogenesis, directing precursors into mineralocorticoids, glucocorticoids, or sex steroids. Although the 17α-hydroxylase and 17,20-lyase activities of this dual function enzyme have been investigated extensively, until recently no CYP17A1 structures were available to inform our understanding. Structures of CYP17A1 with a range of steroidal inhibitors and substrates are now available. This review relates functional knowledge of this enzyme to structural features defining the selective differentiation between its various substrates. While both hydroxylase and lyase substrates have similar orientations with respect to the heme, subtle differences in hydrogen bonding between CYP17A1 and the C3 substituent at the opposite end of ligands appear to correlate with differential substrate utilization and product formation. Complementary structural information from solution NMR supports cytochrome b allosteric modulation of the lyase reaction, implicating regions involved in ligand access to the otherwise buried active site.
Topics: Animals; Humans; Hydroxylation; Pregnenolone; Progesterone; Stereoisomerism; Steroid 17-alpha-Hydroxylase; Substrate Specificity
PubMed: 27566228
DOI: 10.1016/j.mce.2016.08.035 -
Nature Plants Oct 2023Cardenolides are specialized, steroidal metabolites produced in a wide array of plant families. Cardenolides play protective roles in plants, but these molecules,...
Cardenolides are specialized, steroidal metabolites produced in a wide array of plant families. Cardenolides play protective roles in plants, but these molecules, including digoxin from foxglove (Digitalis spp.), are better known for treatment of congenital heart failure, atrial arrhythmia, various cancers and other chronic diseases. However, it is still unknown how plants synthesize 'high-value', complex cardenolide structures from, presumably, a sterol precursor. Here we identify two cytochrome P450, family 87, subfamily A (CYP87A) enzymes that act on both cholesterol and phytosterols (campesterol and β-sitosterol) to form pregnenolone, the first committed step in cardenolide biosynthesis in the two phylogenetically distant plants Digitalis purpurea and Calotropis procera. Arabidopsis plants overexpressing these CYP87A enzymes ectopically accumulated pregnenolone, whereas silencing of CYP87A in D. purpurea leaves by RNA interference resulted in substantial reduction of pregnenolone and cardenolides. Our work uncovers the key entry point to the cardenolide pathway, and expands the toolbox for sustainable production of high-value plant steroids via synthetic biology.
Topics: Cardenolides; Plants; Digitalis; Pregnenolone
PubMed: 37723202
DOI: 10.1038/s41477-023-01515-9 -
European Journal of Pharmacology Sep 2010Neurosteroids hold great promise for the treatment of diseases of the central nervous system (CNS). We compared the uptake by 11 brain regions and appearance in blood of... (Comparative Study)
Comparative Study
Neurosteroids hold great promise for the treatment of diseases of the central nervous system (CNS). We compared the uptake by 11 brain regions and appearance in blood of tritium-labeled pregnenolone and progesterone after intranasal and intravenous (IV) injection. Both neurosteroids appeared in blood and brain after either method of administration, but with important differences in uptake. Bioavailability based on appearance in arterial serum showed that about 23% and 14% of the intranasal administered doses of pregnenolone and progesterone, respectively, entered the blood. Brain levels were about two fold lower after intranasal administration for the two neurosteroids. With intranasal administration, brain levels of the two steroids did not vary over time (2-120 min), whereas brain levels were higher early (10 min or less) after i.v. administration. With i.v. administration, uptake by brain regions did not vary, whereas the olfactory bulb, hippocampus, and hypothalamus had high uptake rates after intranasal administration. Intranasal administration of prenenolone improved memory, whereas progesterone decreased anxiety, thus demonstrating that therapeutic levels of neurosteroids can be delivered to the brain by intranasal administration. The neurosteroids were rapidly degraded after i.v. or intranasal delivery, but pregnenolone was more resistant to degradation in the brain after intranasal administration and in serum after i.v. administration. These results show that either the i.v. or intranasal routes of administration can deliver neurosteroids to blood and brain, but that the two routes have significant differences with intranasal administration favoring some brain regions.
Topics: Administration, Intranasal; Animals; Biological Availability; Biological Transport; Brain; Central Nervous System; Hippocampus; Hypothalamus; Injections, Intravenous; Male; Mice; Neurotransmitter Agents; Pregnenolone; Progesterone
PubMed: 20570588
DOI: 10.1016/j.ejphar.2010.05.033 -
Journal of Inorganic Biochemistry Mar 2023Cytochrome P450 17A1 (CYP17A1) catalyzes 17α-hydroxylation and 17,20-lyase reactions with steroid hormones. Mice contain an orthologous Cyp17a1 enzyme in the genome,...
Cytochrome P450 17A1 (CYP17A1) catalyzes 17α-hydroxylation and 17,20-lyase reactions with steroid hormones. Mice contain an orthologous Cyp17a1 enzyme in the genome, and its amino acid sequence has high similarity with human CYP17A1. We purified recombinant mouse Cyp17a1 and characterized its oxidation reactions with progesterone and pregnenolone. The open reading frame of the mouse Cyp17a1 gene was inserted and successfully expressed in Escherichia coli and then purified using Ni-nitrilotriacetic acid (NTA) affinity column chromatography. Purified mouse Cyp17a1 displayed typical Type I binding titration spectral changes upon the addition of progesterone, 17α-OH progesterone, pregnenolone, and 17α-OH pregnenolone, with similar binding affinities to those of human CYP17A1. Catalytic activities for 17α-hydroxylation and 17,20-lyase reactions were studied using ultra-performance liquid chromatography (UPLC)-mass spectrometry analysis. Mouse Cyp17a1 showed cytochrome b stimulation in catalysis. In comparison to human enzyme, much higher specificity constants (k/K) were observed with mouse Cyp17a1. In the reactions of Δ4-steroids (progesterone and 17α-OH progesterone), the specificity constants were 2100 times higher than the human enzyme. The addition of cytochrome b produced significant stimulation of 17,20-lyase activities of mouse Cyp17a1. Two Arg mutants of mouse Cyp17a1 (R347H and R358Q) displayed a larger decrease in 17,20-lyase reaction (from 17α-OH pregnenolone to dehydroepiandrosterone, DHEA) than 17α-hydroxylation, indicating that -as in human CYP17A1-these basic residues in mouse Cyp17a1 are important in interacting with the cytochrome b protein in the lyase reactions.
Topics: Humans; Mice; Animals; Progesterone; Steroid 17-alpha-Hydroxylase; Lyases; Cytochromes b; Hydroxylation; Steroids; Pregnenolone; Catalysis
PubMed: 36640554
DOI: 10.1016/j.jinorgbio.2022.112085 -
International Journal of Molecular... Aug 2019CDKL5 deficiency disorder (CDD) is a severe neurodevelopmental encephalopathy caused by mutations in the X-linked gene that encodes a serine/threonine kinase. CDD is... (Review)
Review
CDKL5 deficiency disorder (CDD) is a severe neurodevelopmental encephalopathy caused by mutations in the X-linked gene that encodes a serine/threonine kinase. CDD is characterised by the early onset of seizures and impaired cognitive and motor skills. Loss of CDKL5 in vitro and in vivo affects neuronal morphology at early and late stages of maturation, suggesting a link between CDKL5 and the neuronal cytoskeleton. Recently, various microtubule (MT)-binding proteins have been identified as interactors of CDKL5, indicating that its roles converge on regulating MT functioning. MTs are dynamic structures that are important for neuronal morphology, migration and polarity. The delicate control of MT dynamics is fundamental for proper neuronal functions, as evidenced by the fact that aberrant MT dynamics are involved in various neurological disorders. In this review, we highlight the link between CDKL5 and MTs, discussing how CDKL5 deficiency may lead to deranged neuronal functions through aberrant MT dynamics. Finally, we discuss whether the regulation of MT dynamics through microtubule-targeting agents may represent a novel strategy for future pharmacological approaches in the CDD field.
Topics: Animals; Epileptic Syndromes; Humans; Microtubules; Neurons; Pregnenolone; Spasms, Infantile
PubMed: 31438497
DOI: 10.3390/ijms20174075 -
Journal of Neurophysiology Jan 2021Vagal afferent fibers contact neurons in the nucleus of the solitary tract (NTS) and release glutamate via three distinct release pathways: synchronous, asynchronous,...
Vagal afferent fibers contact neurons in the nucleus of the solitary tract (NTS) and release glutamate via three distinct release pathways: synchronous, asynchronous, and spontaneous. The presence of TRPV1 in vagal afferents is predictive of activity-dependent asynchronous glutamate release along with temperature-sensitive spontaneous vesicle fusion. However, pharmacological blockade or genetic deletion of TRPV1 does not eliminate the asynchronous profile and only attenuates the temperature-dependent spontaneous release at high temperatures (>40°C), indicating additional temperature-sensitive calcium conductance(s) contributing to these release pathways. The transient receptor potential cation channel melastatin subtype 3 (TRPM3) is a calcium-selective channel that functions as a thermosensor (30-37°C) in somatic primary afferent neurons. We predict that TRPM3 is expressed in vagal afferent neurons and contributes to asynchronous and spontaneous glutamate release pathways. We investigated these hypotheses via measurements on cultured nodose neurons and in brainstem slice preparations containing vagal afferent to NTS synaptic contacts. We found histological and genetic evidence that TRPM3 is highly expressed in vagal afferent neurons. The TRPM3-selective agonist, pregnenolone sulfate, rapidly and reversibly activated the majority (∼70%) of nodose neurons; most of which also contained TRPV1. We confirmed the role of TRPM3 with pharmacological blockade and genetic deletion. In the brain, TRPM3 signaling strongly controlled both basal and temperature-driven spontaneous glutamate release. Surprisingly, genetic deletion of TRPM3 did not alter synchronous or asynchronous glutamate release. These results provide convergent evidence that vagal afferents express functional TRPM3 that serves as an additional temperature-sensitive calcium conductance involved in controlling spontaneous glutamate release onto neurons in the NTS. Vagal afferent signaling coordinates autonomic reflex function and informs associated behaviors. Thermosensitive transient receptor potential (TRP) channels detect temperature and nociceptive stimuli in somatosensory afferent neurons, however their role in vagal signaling remains less well understood. We report that the TRPM3 ion channel provides a major thermosensitive point of control over vagal signaling and synaptic transmission. We conclude that TRPM3 translates physiological changes in temperature to neurophysiological outputs and can serve as a cellular integrator in vagal afferent signaling.
Topics: Action Potentials; Animals; Excitatory Postsynaptic Potentials; Exocytosis; Glutamic Acid; Hot Temperature; Male; Neurons, Afferent; Pregnenolone; Rats; Rats, Sprague-Dawley; TRPM Cation Channels; Vagus Nerve
PubMed: 33296617
DOI: 10.1152/jn.00229.2020 -
Environment International Jul 2022Hormones play critical roles in facilitating pregnancy progression and the onset of parturition. Several classes of environmental contaminants, including fine...
Hormones play critical roles in facilitating pregnancy progression and the onset of parturition. Several classes of environmental contaminants, including fine particulate matter (PM) and ambient temperature, have been shown to alter hormone biosynthesis or activity. However, epidemiologic research has not considered PM in relation to a broader range of steroid hormones, particularly in pregnant women. Using metabolomics data collected within 20-40 weeks of gestation in an ethnically diverse pregnancy cohort study, we identified 42 steroid hormones that we grouped into five classes (pregnenolone, androgens, estrogens, progestin, and corticosteroids) based on their biosynthesis type. We found that exposure to PM during the pre-conception and early prenatal periods was associated with higher maternal androgen concentrations in late pregnancy. We also detected a positive association between early pregnancy PM exposure and maternal pregnenolone levels and a marginal positive association between early pregnancy PM exposure and progestin levels. When considering each hormone metabolite individually, we found positive associations between early pregnancy PM exposure and five steroids, two of which survived multiple comparison testing: 11beta-hydroxyandrosterone glucuronide (a pregnenolone steroid) and adrosteroneglucuronide (a progestin steroid). None of the steroid classes were statistically significant associated with ambient temperature. In sex-stratified analyses, we did not detect any sex differences in our associations. This is the first study showing that exposure to fine particulate matter during the pre-conception and early prenatal periods can lead to altered steroid adaptation during the state of pregnancy, which has been shown to have potential consequences on maternal and child health.
Topics: Air Pollutants; Air Pollution; Child; Cohort Studies; Female; Humans; Male; Maternal Exposure; Particulate Matter; Pregnancy; Pregnenolone; Progestins; Steroids; Temperature
PubMed: 35700570
DOI: 10.1016/j.envint.2022.107320 -
British Journal of Anaesthesia Mar 1997We have studied the interaction of pregnenolone sulphate and pregnenolone with 5 beta-pregnanolone- and hexobarbitone-induced anaesthesia in male rats using an EEG...
We have studied the interaction of pregnenolone sulphate and pregnenolone with 5 beta-pregnanolone- and hexobarbitone-induced anaesthesia in male rats using an EEG threshold method. Burst suppression of the EEG of 1 s or more ("silent second" (SS)), was used as a criterion of deep anaesthesia. The effects of the steroid solvents albumin and beta-cyclodextrin were assessed by dose-response curves. Despite a significant increase in hexobarbitone threshold dose in relation to increased doses of albumin, there was no correlation between albumin dose and hexobarbitone concentrations in serum, fat and brain tissues. There was no significant difference in threshold concentrations of hexobarbitone between controls given albumin and those pretreated with pregnenolone. In subsequent experiments, 20% beta-cyclodextrin was used as steroid solvent and its volume was maintained at less than 3.0 ml kg-1 during pretreatment. Neither pregnenolone sulphate nor pregnenolone significantly altered the potency of 5 beta-pregnanolone for induction of anaesthesia. Furthermore, there was no interaction of pregnenolone sulphate and pregnenolone on induction of anaesthesia when hexobarbitone was used for anaesthesia.
Topics: Albumins; Anesthetics, Intravenous; Animals; Dose-Response Relationship, Drug; Drug Interactions; Electroencephalography; Hexobarbital; Male; Pregnanolone; Pregnenolone; Rats; Rats, Sprague-Dawley
PubMed: 9135318
DOI: 10.1093/bja/78.3.328