Jan P. Mattsson

Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis

Osteopetrosis includes a group of inherited diseases in which inadequate bone resorption is caused by osteoclast dysfunction. Although molecular defects have been described for many animal models of osteopetrosis, the gene responsible for most cases of the severe human form of the disease (infantile malignant osteopetrosis) is unknown. Infantile malignant autosomal recessive osteopetrosis (MIM 259700) is a severe bone disease with a fatal outcome, generally within the first decade of life. Osteoclasts are present in normal or elevated numbers in individuals affected by autosomal recessive osteopetrosis, suggesting that the defect is not in osteoclast differentiation, but in a gene involved in the functional capacity of mature osteoclasts. Some of the mouse mutants have a decreased number of osteoclasts, which suggests that the defect directly interferes with osteoclast differentiation. In other mutants, it is the function of the osteoclast that seems to be affected, as they show normal or elevated numbers of non-functioning osteoclasts. Here we show that TCIRG1, encoding the osteoclast-specific 116-kD subunit of the vacuolar proton pump, is mutated in five of nine patients with a diagnosis of infantile malignant osteopetrosis. Our data indicate that mutations in TCIRG1 are a frequent cause of autosomal recessive osteopetrosis in humans.

Omeprazole and bafilomycin, two proton pump inhibitors: differentiation of their effects on gastric, kidney and bone H(+)-translocating ATPases.

The effects of omeprazole and bafilomycin on processes dependent on two different types of H(+)-translocating ATPases were compared. A H(+)-ATPase of the E1E2-type, the H+,K(+)-ATPase, was purified from gastric mucosa. Vacuolar type H(+)-ATPases were prepared both from kidney medulla and from osteoclast-containing medullary bone. H+,K(+)-ATPase-mediated proton transport in gastric vesicles was selectively inhibited by omeprazole with a high potency (inhibitory concentrations greater than or equal to 3 microM) and in time- and pH-dependent manner. This result is consistent with the mechanism of action of omeprazole, which is dependent on acid-induced transformation of the drug into an active inhibitor reacting with luminally accessible sulfhydryl groups of the enzyme. Accordingly, the presence of the membrane-impermeable mercaptane glutathione did not affect the inhibitory action of omeprazole on the H+,K(+)-ATPase. Proton transport in kidney- and bone-derived membrane vesicles was also inhibited by omeprazole, but with a lower potency (inhibitory concentrations greater than or equal to 100 microM). Furthermore, the presence of glutathione totally abolished this inhibition, indicating that cytosolic, rather than luminal, SH-groups of the respective vacuolar H(+)-ATPase were interacting with omeprazole at high concentrations. In line with these results, it was found that omeprazole was much more potent in inhibiting acid production in isolated gastric glands (IC50 approximately 0.25 microM) than in inhibiting osteoclast-mediated 45Ca-release in isolated mouse calvaria (IC50 approximately 200 microM). Bafilomycin, on the other hand, was much more effective in inhibiting proton transport mediated by the vacuolar H(+)-ATPases in the kidney- and bone-derived membrane vesicles (IC50 approximately 2 nM) than in inhibiting H+,K(+)-ATPase-mediated proton transport in gastric membrane vesicles (IC50 approximately 50 microM). Thus, approximately 10(4) times higher concentrations of bafilomycin were needed to inhibit the H+,K(+)-ATPase to the same extent as the vacuolar H(+)-ATPase. A similar difference in potency of bafilomycin was found when its inhibitory effect was determined in isolated mouse calvaria (IC50 approximately 2.5 nM) and in isolated gastric glands (IC50 approximately 5 microM). Hence, omeprazole was found to be a specific inhibitor of the H+,K(+)-ATPase under physiological conditions, i.e. in the presence of glutathione, while bafilomycin was found to be selective towards vacuolar H(+)-ATPases.

Effects of GABA agonists on body temperature regulation in GABAB(1)−/− mice

Activation of GABAB receptors evokes hypothermia in wildtype (GABAB(1)+/+) but not in GABAB receptor knockout (GABAB(1)−/−) mice. The aim of the present study was to determine the hypothermic and behavioural effects of the putative GABAB receptor agonist γ‐hydroxybutyrate (GHB), and of the GABAA receptor agonist muscimol. In addition, basal body temperature was determined in GABAB(1)+/+, GABAB(1)+/− and GABAB(1)−/− mice. GABAB(1)−/− mice were generated by homologous recombination in embryonic stem cells. Correct gene targeting was assessed by Southern blotting, PCR and Western blotting. GABAB receptor‐binding sites were quantified with radioligand binding. Measurement of body temperature was done using subcutaneous temperature‐sensitive chips, and behavioural changes after drug administration were scored according to a semiquantitative scale. GABAB(1)−/− mice had a short lifespan, probably caused by generalised seizure activity. No histopathological or blood chemistry changes were seen, but the expression of GABAB(2) receptor protein was below the detection limit in brains from GABAB(1)−/− mice, in the absence of changes in mRNA levels. GABAB receptor‐binding sites were absent in brain membranes from GABAB(1)−/− mice. GABAB(1)−/− mice were hypothermic by approximately 1°C compared to GABAB(1)+/+ and GABAB(1)+/− mice. Injection of baclofen (9.6 mg kg−1) produced a large reduction in body temperature and behavioural effects in GABAB(1)+/+ and in GABAB(1)+/− mice, but GABAB(1)−/− mice were unaffected. The same pattern was seen after administration of GHB (400 mg kg−1). The GABAA receptor agonist muscimol (2 mg kg−1), on the other hand, produced a more pronounced hypothermia in GABAB(1)−/−mice. In GABAB(1)+/+ and GABAB(1)+/− mice, muscimol induced sedation and reduced locomotor activity. However, when given to GABAB(1)−/− mice, muscimol triggered periods of intense jumping and wild running. It is concluded that hypothermia should be added to the characteristics of the GABAB(1)−/−phenotype. Using this model, GHB was shown to be a selective GABAB receptor agonist. In addition, GABAB(1)−/− mice are hypersensitive to GABAA receptor stimulation, indicating that GABAB tone normally balances GABAA‐mediated effects.

Differential regulation of TRP channels in a rat model of neuropathic pain.

ABSTRACT Neuropathic pain is a chronic disease resulting from dysfunction of the nervous system often due to peripheral nerve injury. Hypersensitivity to sensory stimuli (mechanical, thermal or chemical) is a common source of pain in patients and ion channels involved in detecting these stimuli are possible candidates for inducing and/or maintaining the pain. Transient receptor potential (TRP) channels expressed on nociceptors respond to different sensory stimuli and a few of them have been studied previously in the models of neuropathic pain. Using real‐time PCR for quantification of all known TRP channels we identified several TRP channels, which have not been associated with nociception or neuropathic pain before, to be expressed in the DRG and to be differentially regulated after spared nerve injury (SNI). Of all TRP channel members, TRPML3 showed the most dramatic change in animals exhibiting neuropathic pain behaviour compared to control animals. In situ hybridisation showed a widespread increase of expression in neurons of small, medium and large cell sizes, indicating expression in multiple subtypes. Co‐localisation of TRPML3 with CGRP, NF200 and IB4 staining confirmed a broad subtype distribution. Expression studies during development showed that TRPML3 is an embryonic channel that is induced upon nerve injury in three different nerve injury models investigated. Thus, the current results link for the first time a re‐expression of TRPML3 with the development of neuropathic pain conditions. In addition, decreased mRNA levels after SNI were seen for TRPM6, TRPM8, TRPV1, TRPA1, TRPC3, TRPC4 and TRPC5.

Cellular subtype distribution and developmental regulation of TRPC channel members in the mouse dorsal root ganglion

Transient receptor potential (TRP) channels play essential roles in sensory physiology and their expression in different classes of sensory neurons reflect distinct receptive properties of these neurons. While expression of the TRPV, TRPA, and to a certain degree TRPM classes of channels has been studied in sensory neurons, little is known about the expression and regulation of TRPC channels. In this study we examined the regulation of all TRPC members (TRPC1–C7) throughout embryonic and postnatal development of the dorsal root ganglion (DRG) and nodose ganglion (NG). In adult mice, mRNAs for all channels were present in the DRG, with TRPC1, 3, and 6 being the most abundant, TRPC2, C4, and C5 at lower levels, and TRPC7 at very low levels. While TRPC2 mRNAs were downregulated from high levels at embryonic (E) day 12 and E14 until adult, TRPC4, C5, and C7 expressions increased from E12 to peak levels at E18. TRPC1, C3, and C6, the most abundant TRPC channel mRNAs, increased progressively from E12 to adult. Expression and regulation of TRPC channels mRNAs in the NG were unexpectedly similar to the DRG. TRPC1 and C2 was expressed in the neurofilament‐200 (NF‐200)‐positive large size subclass of neurons, while TRPC3 mRNAs expression, which stained up to 35% of DRG neurons, was almost exclusively present in nonpeptidergic isolectin B4 (IB4)‐positive small size neurons that were largely TRPV1‐negative. Our results suggest important roles of the TRPC family of channels in sensory physiology of both nociceptive as well as nonnociceptive classes of neurons. J. Comp. Neurol. 503:35–46, 2007.

Dynamic expression of the TRPM subgroup of ion channels in developing mouse sensory neurons

Despite the significance of transient receptor potential (TRP) channels in sensory physiology, little is known of the expression and developmental regulation of the TRPM (melastatin) subgroup in sensory neurons. In order to find out if the eight TRPM subgroup members (TRPM1-TRPM8) have a possible role in the sensory nervous system, we characterized the developmental regulation of their expression in mouse dorsal root ganglion (DRG) from embryonic (E) day 12 to adulthood. Transcripts for all channels except for TRPM1 were detected in lumbar and thoracic DRG and in nodose ganglion (NG) with distinguishable expression patterns from E12 until adult. For most channels, the expression increased from E14 to adult with the exception of TRPM5, which displayed transient high levels during embryonic and early postnatal stages. Cellular localization of TRPM8 mRNA was found only in a limited subset of very small diameter neurons distinct in size from other populations. These neurons did not bind isolectin B4 (IB4) and expressed neither the neuropeptide calcitonin gene-related peptide (CGRP) nor neurofilament (NF)200. This suggests that TRPM8(+) thermoreceptive sensory neurons fall into a separate group of very small sized neurons distinct from peptidergic and IB4(+) subtypes of sensory neurons. Our results, showing the expression and dynamic regulation of TRPM channels during development, indicate that many TRPM subfamily members could participate during nervous system development and in the adult by determining distinct physiological properties of sensory neurons.

(R)-(3-amino-2-fluoropropyl) phosphinic acid (AZD3355), a novel GABAB receptor agonist, inhibits transient lower esophageal sphincter relaxation through a peripheral mode of action.

Gastroesophageal reflux disease (GERD) affects >10% of the Western population. Conventionally, GERD is treated by reducing gastric acid secretion, which is effective in most patients but inadequate in a significant minority. We describe a new therapeutic approach for GERD, based on inhibition of transient lower esophageal sphincter relaxation (TLESR) with a proposed peripherally acting GABAB receptor agonist, (R)-(3-amino-2-fluoropropyl)phosphinic acid (AZD3355). AZD3355 potently stimulated recombinant human GABAB receptors and inhibited TLESR in dogs, with a biphasic dose-response curve. In mice, AZD3355 produced considerably less central side effects than the prototypical GABAB receptor agonist baclofen but evoked hypothermia at very high doses (blocked by a GABAB receptor antagonist and absent in GABAB−/− mice). AZD3355 and baclofen differed markedly in their distribution in rat brain; AZD3355, but not baclofen, was concentrated in circumventricular organs as a result of active uptake (shown by avid intracellular sequestration) and related to binding of AZD3355 to native GABA transporters in rat cerebrocortical membranes. AZD3355 was also shown to be transported by all four recombinant human GABA transporters. AR-H061719 [(R/S)-(3-amino-2-fluoropropyl)phosphinic acid], (the racemate of AZD3355) inhibited the response of ferret mechanoreceptors to gastric distension, further supporting its peripheral site of action on TLESR. In summary, AZD3355 probably inhibits TLESR through stimulation of peripheral GABAB receptors and may offer a potential new approach to treatment of GERD.

Recent Advances in Non-Competitive mGlu5 Receptor Antagonists and their Potential Therapeutic Applications

Extensive research into the functions of glutamate and glutamate receptors in the central nervous system (CNS) has shown an essential role of metabotropic glutamate (mGlu) receptors in normal brain functions, but also in neurological and psychiatric disorders. The precise functions of these receptors remain undefined, and progress toward understanding their functions has been hampered by the lack of selective ligands with appropriate pharmacokinetic properties. The Group I mGlu receptor, mGlu5, is well positioned to regulate and fine-tune neuronal excitability and synaptic transmission through its modulation of various signal transduction pathways and interactions with other transmitter systems. Therefore, the mGlu5 receptor may be an important therapeutic target for the treatment of disorders of the central nervous system. The discovery of MPEP 3, a non-competitive mGlu5 receptor antagonist, provided a potent, selective, systemically active tool compound for proof of concept studies in animal models of various disease states. These studies have led to greater understanding of possible therapeutic applications of mGlu5 receptor antagonists in recent years, suggesting their use in a number of disease states, including chronic pain, various psychiatric and neurological disorders, substance abuse and withdrawal, obesity and gastroesophageal reflux disease (GERD). Together, these findings have intensified efforts to find other non-competitive mGlu5 receptor antagonists and have led to the discovery of several second-generation compounds, a few of which are in preclinical evaluations. There have been several recent reviews on mGlu receptor. This article highlights recent efforts on the design, synthesis and development of novel, non-competitive mGlu5 receptor antagonists and studies to understand their in vitro mechanisms of action and in vivo pharmacological profiles. Emphasis is also given to recent advances in the potential therapeutic applications of non-competitive mGlu5 receptor antagonists.

Synthesis and Pharmacological Evaluation of Novel γ-Aminobutyric Acid Type B (GABAB) Receptor Agonists as Gastroesophageal Reflux Inhibitors

We have previously demonstrated that the prototypical GABA B receptor agonist baclofen inhibits transient lower esophageal sphincter relaxations (TLESRs), the most important mechanism for gastroesophageal reflux. Thus, GABA B agonists could be exploited for the treatment of gastroesophageal reflux disease. However, baclofen, which is used as an antispastic agent, and other previously known GABA B agonists can produce CNS side effects such as sedation, dizziness, nausea, and vomiting at higher doses. We now report the discovery of atypical GABA B agonists devoid of classical GABA B agonist related CNS side effects at therapeutic doses and the optimization of this type of compound for inhibition of TLESRs, which has resulted in a candidate drug ( R)- 7 (AZD3355) that is presently being evaluated in man.

Effects of repeated administration of baclofen to rats on GABAB receptor binding sites and subunit expression in the brain.

Repeated stimulation of the GABAB receptor with baclofen frequently produces tolerance, the underlying mechanisms of which are poorly understood. The purpose of the present work was to determine whether repeated administration of baclofen to rats is accompanied by changes in cerebral GABAB receptor binding sites, mRNA for the subunits GABAB(1) and GABAB(2), and protein levels for these subunits. Rats were injected with placebo or baclofen (20 μmmol/kg subcutaneously) once daily for 14 days. Decreases in body temperature were measured as an index of pharmacological effects of baclofen. Binding of radiolabeled GABA to GABAB receptors was quantitated in brain membranes, mRNA levels were determined using quantitative real-time PCR, and GABAB receptor protein levels were assessed with Western blot analysis. Baclofen caused a decline in temperature amounting to approximately 2.5 °C after the first dose. This effect was partly lost after the fifth and abolished after the seventh injection. Despite the complete development of tolerance, there were no significant alterations in GABAB receptor binding sites (number or affinity) or mRNA levels for the subtypes GABAB(1a), GABAB(1b), or GABAB(2). Receptor protein levels were also unchanged. It is concluded that baclofen induces tolerance through mechanisms other than down-regulation of GABAB receptor transcription or translation.