Martin Kohler

Relationship between sodium channel NaV1.3 expression and neuropathic pain behavior in rats

&NA; A multitude of voltage‐gated sodium channel subtypes (NaV1) are expressed in primary sensory neurons where they influence excitability via their role in the generation and propagation of action potentials. Peripheral nerve injury alters the expression of several NaV1subtypes, but among these only NaV1.3 is up‐regulated in dorsal root ganglia (DRG) neurons. The increased expression of NaV1.3 implicates this subtype in the development and maintenance of neuropathic pain, but its contribution to neuropathic pain behavior has not been examined. Using the spared nerve injury (SNI) model, we found that peripheral nerve lesion increased NaV1.3‐like immunoreactivity (–LI) in DRG neurons and that mechanical allodynia was partially alleviated following oral administration of two NaV1 blockers, mexiletine (30 and 100 mg/kg, p.o.) and lamotrigine (30 and 100 mg/kg, p.o.). Intrathecal administration of antisense oligonucleotides (4 days) selective for NaV1.3 decreased NaV1.3 immunostaining in the DRG by 50% in the SNI model, but did not attenuate mechanical or cold allodynia. Moreover, we found that only 18% of NaV1.3 positive neurons also expressed activated transcription factor‐3 (ATF3), a marker of injured neurons. We then selectively axotomized a cutaneous nerve (sural) and a muscle nerve (gastrocnemius) in order to identify if NaV1.3 up‐regulation is dependent on cutaneous and/or muscle afferent activation and found that the numbers of neurons expressing NaV1.3 was proportional to the magnitude of the injury, but independent of the nature of innervation. These results suggest that NaV1.3 increases in primary sensory neurons that are not directly damaged in response to injury. Thus, although NaV1.3 is up‐regulated in a subpopulation of DRG neurons after injury, reduction in the expression of NaV1.3 subtype alone is not sufficient to influence the NaV1‐dependent behavioral hypersensitivity associated with nerve injury.

Somatostatin Receptor Subtypes 2 and 5 Inhibit Corticotropin-Releasing Hormone-Stimulated Adrenocorticotropin Secretion from AtT-20 Cells

Somatostatin (SRIH) regulates pituitary adrenocorticotropin (ACTH) secretion by interacting with a family of homologous G protein-coupled membrane receptors. The SRIH receptor subtypes (sst₁–sst₅) that control ACTH release remain unknown. Using novel, subtype-selective SRIH analogs, we have identified the SRIH receptor subtypes involved in regulating ACTH release from AtT-20 cells, a model for cell line pituitary corticotropes. Radioligand-binding studies with ¹²⁵I-SRIH-14 and ¹²⁵I-SRIH-28 showed that SRIH-14 and SRIH-28 recognized specific, high-affinity and saturable membrane-binding sites. Nonpeptidyl agonists with selectivity for the sst₂ (L-779,976; compound 2) or sst₁/sst₅) (L-817,818; compound 5) receptor subtypes potently displaced ¹²⁵I-SRIH-28 from AtT-20 cell membranes, while agonists selective for the sst₁ (L-779,591; compound 1), sst₃ (L-796,778; compound 3) or sst₄ (L-803,087; compound 4) subtypes were inactive. Tyr¹¹-SRIH-14, compound 2 (sst₂) or compound 5 (sst₅) inhibited forskolin and corticotropin-releasing hormone (CRH)-induced increases in intracellular cAMP. Furthermore, the sst₂ and sst₅ agonists potently inhibited CRH-induced ACTH release from AtT-20 cells. These results provide the first evidence that sst₂ and sst₅ receptor subtypes, but not sst₁, sst₃ or sst₄, inhibit cAMP accumulation and regulate ACTH secretion in the AtT-20 cell model of the rodent corticotrope.

The behavioral and neuroanatomical effects of IB4-saporin treatment in rat models of nociceptive and neuropathic pain.

One distinguishing feature of primary afferent neurons is their ability to bind the lectin IB(4). Previous work suggested that neurons in the inner part of lamina II (IIi), onto which IB(4)-positive sensory neurons project, facilitate nociceptive transmission following tissue or nerve injury. Using an IB(4)-saporin conjugate (IB(4)-SAP), we examined the contribution of IB(4)-positive neurons to nociceptive processing in rats with and without nerve injury. Intrasciatic injection of IB(4)-SAP (5 mug/5 mul) significantly decreased IB(4)-labeling and immunoreactive P(2)X(3) in the spinal cord and delayed the behavioral and neuroanatomical consequences of L5 spinal nerve ligation (SNL) injury. In the absence of injury, thermal and mechanical nociceptive thresholds increased 2 weeks post-treatment only in IB(4)-SAP-treated, but not control (saline or saporin only), rats. Acute NGF-induced hyperalgesia was also attenuated following IB(4)-SAP treatment. In the SNL model, mechanical allodynia failed to develop 1 and 2 weeks post-injury, but was fully established by 4 weeks. Moreover, neuropeptide Y immunoreactivity (NPY-ir), which increases in the spinal cord after nerve injury, was unchanged in IB(4)-SAP-treated animals whereas immunoreactive PKCgamma decreased 2, but not 4, weeks post-injury. Quantitative RT-PCR revealed a reduction in P(2)X(3) mRNA in L4 DRG of IB(4)-SAP-treated animals, but no change in TrkA expression. Our results suggest that IB(4)-positive neurons in L4 are required for the full expression of NGF-induced hyperalgesia and participate in the behavioral and anatomical consequences that follow injury to the L5 spinal nerve.

Biophysical and pharmacological properties of the voltage-gated potassium current of human pancreatic β-cells

Voltage‐gated potassium (Kv) currents of human pancreatic islet cells were studied by whole‐cell patch clamp recording. On average, 75% of the cells tested were identified as β‐cells by single cell, post‐recording RT‐PCR for insulin mRNA. In most cells, the dominant Kv current was a delayed rectifier. The delayed rectifier activated at potentials above −20 mV and had a V½ for activation of −5.3 mV. Onset of inactivation was slow for a major component (τ= 3.2 s at +20 mV) observed in all cells; a smaller component (τ= 0.30 s) with an amplitude of ∼25% was seen in some cells. Recovery from inactivation had a τ of 2.5 s at −80 mV and steady‐state inactivation had a V½ of −39 mV. In 12% of cells (21/182) a low‐threshold, transient Kv current (A‐current) was present. The A‐current activated at membrane potentials above −40 mV, inactivated with a time constant of 18.5 ms at −20 mV, and had a V½ for steady‐state inactivation of −52 mV. TEA inhibited total Kv current with an IC50= 0.54 mm and PAC, a disubstituted cyclohexyl Kv channel inhibitor, inhibited with an IC50= 0.57 μm. The total Kv current was insensitive to margatoxin (100 nm), agitoxin‐2 (50 nm), kaliotoxin (50 nm) and ShK (50 nm). Hanatoxin (100 nm) inhibited total Kv current by 65% at +20 mV. Taken together, these data provide evidence of at least two distinct types of Kv channels in human pancreatic β‐cells and suggest that more than one type of Kv channel may be involved in the regulation of glucose‐dependent insulin secretion.

The design and synthesis of novel spirocyclic heterocyclic sulfone ROMK inhibitors as diuretics

A spirocyclic class of ROMK inhibitors was developed containing a structurally diverse heterocyclic sulfone moiety and spirocyclic core starting from lead 1. These compounds not only displayed exquisite ROMK potency but significantly improved selectivity over hERG. The lead compounds were found to have favorable pharmacokinetic properties and displayed robust diuretic, natriuretic and blood pressure lowering effects in spontaneously hypertensive rats.

Characteristic time courses of cortical and medullary sodium signals measured by noninvasive 23Na-MRI in rat kidney induced by furosemide

To characterize regional kidney sodium response by MRI following NKCC2 inhibition.