Leigh B. MacMillan

Central Hypotensive Effects of the α2a-Adrenergic Receptor Subtype

α2-Adrenergic receptors (α2ARs) present in the brainstem decrease blood pressure and are targets for clinically effective antihypertensive drugs. The existence of three α2AR subtypes, the lack of subtype-specific ligands, and the cross-reactivity of α2AR agonists with imidazoline receptors has precluded an understanding of the role of individual α2AR subtypes in the hypotensive response. Gene targeting was used to introduce a point mutation into the α2aAR subtype in the mouse genome. The hypotensive response to α2AR agonists was lost in the mutant mice, demonstrating that the α2aAR subtype plays a principal role in this response.

The α2a Adrenergic Receptor Subtype Mediates Spinal Analgesia Evoked by α2 Agonists and Is Necessary for Spinal Adrenergic–Opioid Synergy

Agonists acting at α2 adrenergic and opioid receptors have analgesic properties and act synergistically when co-administered in the spinal cord; this synergy may also contribute to the potency and efficacy of spinally administered morphine. The lack of subtype-selective pharmacological agents has previously impeded the definition of the adrenergic receptor subtype(s) mediating these effects. We therefore exploited a genetically modified mouse line expressing a point mutation (D79N) in the α2a adrenergic receptor (α2aAR) to investigate the role of the α2aAR in α2 agonist-evoked analgesia and adrenergic–opioid synergy. In the tail-flick test, intrathecal administration of UK 14,304, a nonsubtype-selective α2AR agonist, had no analgesic effect in D79N mice, whereas the analgesic potency of morphine (intrathecal) in this assay was not affected by the mutation. The mutation also decreased α2-agonist-mediated spinal analgesia and blocked the synergy seen in wild-type mice with both the δ-opioid agonist deltorphin II and the μ-opioid agonist [d-ALA2,N-Me-Phe4,Gly-ol5]-Enkephalin (DAMGO) in the substance P behavioral test. In addition, the potency of spinally administered morphine was decreased in this test, suggesting that activation of descending noradrenergic systems impinging on the α2aAR contributes to morphine-induced spinal inhibition in this model. These results demonstrate that the α2aAR subtype is the primary mediator of α2 adrenergic spinal analgesia and is necessary for analgesic synergy with opioids. Thus, combination therapies targeting the α2aAR and opioid receptors may prove useful in maximizing the analgesic efficacy of opioids while decreasing total dose requirements.

CaM kinase augments cardiac L-type Ca2+ current: a cellular mechanism for long Q-T arrhythmias

Early afterdepolarizations (EAD) caused by L-type Ca2+ current (ICa, L) are thought to initiate long Q-T arrhythmias, but the role of intracellular Ca2+ in these arrhythmias is controversial. Rabbit ventricular myocytes were stimulated with a prolonged EAD-containing action potential-clamp waveform to investigate the role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase) in ICa,L during repolarization. ICa,L was initially augmented, and augmentation was dependent on Ca2+ from the sarcoplasmic reticulum because the augmentation was prevented by ryanodine or thapsigargin. ICa,L augmentation was also dependent on CaM kinase, because it was prevented by dialysis with the inhibitor peptide AC3-I and reconstituted by exogenous constitutively active CaM kinase when Ba2+ was substituted for bath Ca2+. Ultrastructural studies confirmed that endogenous CaM kinase, L-type Ca2+ channels, and ryanodine receptors colocalized near T tubules. EAD induction was significantly reduced in current-clamped cells dialyzed with AC3-I (4/15) compared with cells dialyzed with an inactive control peptide (11/15, P = 0.013). These findings support the hypothesis that EADs are facilitated by CaM kinase.Early afterdepolarizations (EAD) caused by L-type Ca2+ current ( I Ca,L) are thought to initiate long Q-T arrhythmias, but the role of intracellular Ca2+ in these arrhythmias is controversial. Rabbit ventricular myocytes were stimulated with a prolonged EAD-containing action potential-clamp waveform to investigate the role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase) in I Ca,L during repolarization. I Ca,L was initially augmented, and augmentation was dependent on Ca2+ from the sarcoplasmic reticulum because the augmentation was prevented by ryanodine or thapsigargin. I Ca,Laugmentation was also dependent on CaM kinase, because it was prevented by dialysis with the inhibitor peptide AC3-I and reconstituted by exogenous constitutively active CaM kinase when Ba2+ was substituted for bath Ca2+. Ultrastructural studies confirmed that endogenous CaM kinase, L-type Ca2+ channels, and ryanodine receptors colocalized near T tubules. EAD induction was significantly reduced in current-clamped cells dialyzed with AC3-I (4/15) compared with cells dialyzed with an inactive control peptide (11/15, P = 0.013). These findings support the hypothesis that EADs are facilitated by CaM kinase.

Brain Actin-associated Protein Phosphatase 1 Holoenzymes Containing Spinophilin, Neurabin, and Selected Catalytic Subunit Isoforms

We previously characterized PP1bp134 and PP1bp175, two neuronal proteins that bind the protein phosphatase 1 catalytic subunit (PP1). Here we purify from rat brain actin-cytoskeletal extracts PP1A holoenzymes selectively enriched in PP1γ1 over PP1β isoforms and also containing PP1bp134 and PP1bp175. PP1bp134 and PP1bp175 were identified as the synapse-localized F-actin-binding proteins spinophilin (Allen, P. B., Ouimet, C. C., and Greengard, P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9956–9561; Satoh, A., Nakanishi, H., Obaishi, H., Wada, M., Takahashi, K., Satoh, K., Hirao, K., Nishioka, H., Hata, Y., Mizoguchi, A., and Takai, Y. (1998) J. Biol. Chem. 273, 3470–3475) and neurabin (Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H., Matsuura, Y., Mizoguchi, A., and Takai, Y. (1997)J. Cell Biol. 139, 951–961), respectively. Recombinant spinophilin and neurabin interacted with endogenous PP1 and also with each other when co-expressed in HEK293 cells. Spinophilin residues 427–470, or homologous neurabin residues 436–479, were sufficient to bind PP1 in gel overlay assays, and selectively bound PP1γ1 from a mixture of brain protein phosphatase catalytic subunits; additional N- and C-terminal sequences were required for potent inhibition of PP1. Immunoprecipitation of spinophilin or neurabin from crude brain extracts selectively coprecipitated PP1γ1 over PP1β. Moreover, immunoprecipitation of PP1γ1 from brain extracts efficiently coprecipitated spinophilin and neurabin, whereas PP1β immunoprecipitation did not. Thus, PP1A holoenzymes containing spinophilin and/or neurabin target specific neuronal PP1 isoforms, facilitating efficient regulation of synaptic phosphoproteins.

α-Adrenoceptors and vascular regulation: Molecular, pharmacologic and clinical correlates

This manuscript is intended to provide a comprehensive review of the alpha-adrenoceptors (ARs) and their role in vascular regulation. The historical development of the concept of receptors and the division of the alpha-ARs into alpha 1 and alpha 2 subtypes is traced. Emphasis will be placed on current understanding of the specific contribution of discrete alpha 1- and alpha 2-AR subtypes in the regulation of the vasculature, selective agonists and antagonists for these receptors, the second messengers utilized by these receptors, the myoplasmic calcium pathways activated to initiate smooth muscle contraction, as well as the clinical uses of agonists and antagonists that work at these receptors. New information is presented that deals with the molecular aspects of ligand interactions with specific subdomains of these receptors, as well as mRNA distribution and the regulation of alpha 1- and alpha 2-AR gene transcription and translation.

Heterozygous α2A-adrenergic receptor mice unveil unique therapeutic benefits of partial agonists

Genetic manipulation of the α2A-adrenergic receptor (α2A-AR) in mice has revealed the role of this subtype in numerous responses, including agonist-induced hypotension and sedation. Unexpectedly, α2-agonist treatment of mice heterozygous for the α2A-AR (α2A-AR+/−) lowers blood pressure without sedation, indicating that more than 50% of α2A-AR must be activated to evoke sedation. We postulated that partial activation of α2A-AR in wild-type α2A-AR+/+ animals could be achieved with partial agonists, agents with variable ability to couple receptor occupancy to effector activation, and might elicit one versus another pharmacological response. In vitro assays reveal that moxonidine is a partial agonist at α2A-AR. Although moxonidine was developed to preferentially interact with imidazoline binding sites, it requires the α2A-AR to lower blood pressure because we observe no hypotensive response to moxonidine in α2A-AR-null (α2A-AR−/−) mice. Moreover, we observe that moxonidine lowers blood pressure without sedation in wild-type mice, consistent with the above hypothesis regarding partial agonists. Our findings suggest that weak partial agonists can evoke response-selective pathways and might be exploited successfully to achieve α2A-AR pharmacotherapy where concomitant sedation is undesirable, i.e., in treatment of depression or attention deficit hyperactivity disorder, in suppression of epileptogenesis, or enhancement of cognition. Furthermore, rigorous physiological and behavioral assessment of mice heterozygous for particular receptors provides a general strategy for elucidation of pathways that might be selectively activated by partial agonists, thus achieving response-specific therapy.

In Vivo Mutation of the α2A-Adrenergic Receptor by Homologous Recombination Reveals the Role of This Receptor Subtype in Multiple Physiological Processes

Publisher Summary α 2 -Adrenergic receptors ( α 2 ARs) are broadly associated with inhibitory actions of norepinephrine and epinephrine. Studies with drugs designed as α 2 AR agonists and antagonists have shown that α 2 ARs participate in a wide range of central nervous system activities, including central regulation of blood pressure, sedation and analgesia, control of affect, and modulation of pituitary hormone release. In the periphery, α 2 ARs inhibit neurotransmitter release from peripheral nervous system neuronal terminals, inhibit insulin secretion by pancreatic β cells, and participate in the regulation of water and electrolyte balance in the kidney. The α 2 ARs that elicit these varied responses represent a structural and functional family of receptor subtypes defined by pharmacological measurements and molecular cloning. Interest of studies has been to establish a mouse line that expressed a mutant α 2A AR receptor with selectively altered signal transduction capabilities. Studies have shown that mutation of the highly conserved aspartate residue at position 79 in the α 2A AR to asparagine (D79N) results in a receptor that is uncoupled from a single signaling pathway. In this purpose the two-step “hit and run” gene-targeting approach is successfully used in mouse embryonic stem cells to establish a mouse line with this D79N α 2A AR mutation. Such studies reveal that the α 2A AR subtype has a role central to several of the clinically desirable effects of α 2A R agonists, suggesting that these functions cannot be separated by administering subtype-specific drugs. Null mutation of the α 2B AR subtype has revealed that this subtype mediates the increase in blood pressure immediately following α 2A R agonist administration. Thus, drugs that are selective for the α 2A AR subtype relative to the α 2B AR subtype may hold promise as improved antihypertensive agents. Sedative side effects might be eliminated if high-affinity partial agonists are developed, especially if higher receptor occupancy is needed to evoke sedation in contrast to hypotension.

Respiratory Pattern and Hypdxic Ventilatory Response in Mice Functionally Lacking α2a-Adrenergic Receptors

In newborn mammals including man the respiratory pattern undergoes changes during the neonatal period10, 11, 26, 27, 36.These developmental changes are characterized by an increase in the duration of inspiratory time (TI), which in species born after relatively short gestations such as opossum10 and mouse27 can be quite marked. In addition to developmental differences in metabolism, pulmonary mechanics and stimulatory input from the periphery the intrinsic membrane properties of inspiratory neurons may contribute to this lengthening of TI. Spike frequency adaptation and afterhyperpolarization is present as early as postnatal day 1 (P1) in rat hypoglossal motoneurons35 and PO in mouse inspiratory neurons located in the pre-Botzinger complex (PBC)28. Entry of calcium through high-voltage-activated (HVA) calcium channels has been shown to be essential for the generation of the AHP in PBC neurons9, ventral nucleus of the solitary tract29 and hypoglossal motoneurons32, 33 by activating slow-conductance calcium-activated potassium channels29,34. Therefore, the inhibiting modulation of HVA calcium channels during development is a potential mechanism by which TI could be prolonged. α2a adrenergic receptors which are coupled by pertussis toxin sensitive G proteins to various effectors, including inhibition of voltage activated Ca2+ channels20, and have been demonstrated in cardiorespiratory areas of the medulla1, 31 are candidates for this role. In order to evaluate their importance respiratory timing in the postnatal period was examined in mice who functionally lack α2a adrenergic receptors.