Reduced density and altered regulation of rat atrial L-type Ca2+ current in heart failure.

Constitutive regulation by PKA has recently been shown to contribute to L-type Ca2+ current (ICaL) at the ventricular t-tubule in heart failure. Conversely, reduction in constitutive regulation by PKA has been proposed to underlie the downregulation of atrial ICaL in heart failure. The hypothesis that downregulation of atrial ICaL in heart failure involves reduced channel phosphorylation was examined. Anesthetized adult male Wistar rats underwent surgical coronary artery ligation (CAL, N=10) or equivalent sham-operation (Sham, N=12). Left atrial myocytes were isolated ~18 wk postsurgery and whole cell currents recorded (holding potential=-80 mV). ICaL activated by depolarizing pulses to voltages from -40 to +50 mV were normalized to cell capacitance and current density-voltage relations plotted. CAL cell capacitances were ~1.67-fold greater than Sham (P ≤ 0.0001). Maximal ICaL conductance (Gmax ) was downregulated more than 2-fold in CAL vs. Sham myocytes (P < 0.0001). Norepinephrine (1 µmol/l) increased Gmax >50% more effectively in CAL than in Sham so that differences in ICaL density were abolished. Differences between CAL and Sham Gmax were not abolished by calyculin A (100 nmol/l), suggesting that increased protein dephosphorylation did not account for ICaL downregulation. Treatment with either H-89 (10 µmol/l) or AIP (5 µmol/l) had no effect on basal currents in Sham or CAL myocytes, indicating that, in contrast to ventricular myocytes, neither PKA nor CaMKII regulated basal ICaL Expression of the L-type α1C-subunit, protein phosphatases 1 and 2A, and inhibitor-1 proteins was unchanged. In conclusion, reduction in PKA-dependent regulation did not contribute to downregulation of atrial ICaL in heart failure.NEW & NOTEWORTHY Whole cell recording of L-type Ca2+ currents in atrial myocytes from rat hearts subjected to coronary artery ligation compared with those from sham-operated controls reveals marked reduction in current density in heart failure without change in channel subunit expression and associated with altered phosphorylation independent of protein kinase A.

Design and nuclear magnetic resonance (NMR) structure determination of the second extracellular immunoglobulin tyrosine kinase A (TrkAIg2) domain construct for binding site elucidation in drug discovery.

The tyrosine kinase A (TrkA) receptor is a validated therapeutic intervention point for a wide range of conditions. TrkA activation by nerve growth factor (NGF) binding the second extracellular immunoglobulin (TrkAIg2) domain triggers intracellular signaling cascades. In the periphery, this promotes the pain phenotype and, in the brain, cell survival or differentiation. Reproducible structural information and detailed validation of protein-ligand interactions aid drug discovery. However, the isolated TrkAIg2 domain crystallizes as a ß-strand-swapped dimer in the absence of NGF, occluding the binding surface. Here we report the design and structural validation by nuclear magnetic resonance spectroscopy of the first stable, biologically active construct of the TrkAIg2 domain for binding site confirmation. Our structure closely mimics the wild-type fold of TrkAIg2 in complex with NGF ( 1WWW .pdb), and the (1)H-(15)N correlation spectra confirm that both NGF and a competing small molecule interact at the known binding interface in solution.

Stimulation of ICa by basal PKA activity is facilitated by caveolin-3 in cardiac ventricular myocytes.

L-type Ca channels (LTCC), which play a key role in cardiac excitation-contraction coupling, are located predominantly at the transverse (t-) tubules in ventricular myocytes. Caveolae and the protein caveolin-3 (Cav-3) are also present at the t-tubules and have been implicated in localizing a number of signaling molecules, including protein kinase A (PKA) and ß2-adrenoceptors. The present study investigated whether disruption of Cav-3 binding to its endogenous binding partners influenced LTCC activity. Ventricular myocytes were isolated from male Wistar rats and LTCC current (ICa) recorded using the whole-cell patch-clamp technique. Incubation of myocytes with a membrane-permeable peptide representing the scaffolding domain of Cav-3 (C3SD) reduced basal ICa amplitude in intact, but not detubulated, myocytes, and attenuated the stimulatory effects of the ß2-adrenergic agonist zinterol on ICa. The PKA inhibitor H-89 also reduced basal ICa; however, the inhibitory effects of C3SD and H-89 on basal ICa amplitude were not summative. Under control conditions, myocytes stained with antibody against phosphorylated LTCC (pLTCC) displayed a striated pattern, presumably reflecting localization at the t-tubules. Both C3SD and H-89 reduced pLTCC staining at the z-lines but did not affect staining of total LTCC or Cav-3. These data are consistent with the idea that the effects of C3SD and H-89 share a common pathway, which involves PKA and is maximally inhibited by H-89, and suggest that Cav-3 plays an important role in mediating stimulation of ICa at the t-tubules via PKA-induced phosphorylation under basal conditions, and in response to ß2-adrenoceptor stimulation.

Targeting nerve growth factor in pain: what is the therapeutic potential?

Chronic pain presents a huge economic and social burden, with existing treatments largely unable to satisfy medical needs. Recently, studies have shown that nerve growth factor (NGF) is a major mediator of inflammatory and neuropathic pain, providing a new therapeutic target. Although originally discovered as a trophic factor for sympathetic and sensory neurons during development, it now appears that in adults, levels of NGF are elevated in many acute and chronic pain conditions. Furthermore, preclinical animal models of inflammatory and neuropathic pain also show increased NGF levels, while the sequestration of NGF alleviates the associated hyperalgesia. The molecular mechanisms involved are being elucidated. This review briefly examines pain signaling pathways and describes currently available analgesics. It then investigates the approaches taken in targeting NGF-mediated pain. Current options being explored include the development of humanized monoclonal antibodies to NGF or its tyrosine kinase receptor TrkA (also known as neurotrophic tyrosine kinase receptor, type 1 [NTRK1]), and the sequestration of NGF using TrkA domain 5 (TrkAd5), a soluble receptor protein that binds NGF with picomolar affinity. Administration of either antibodies or TrkAd5 has been shown to be effective in a number of preclinical models of pain, including cystitis, osteoarthritis, UV irradiation (sunburn), and skeletal bone pain due to fracture or cancer. Other possible future therapies examined in this review include small-molecule TrkA antagonists, which target either the extracellular NGF binding domain of TrkA or its intracellular tyrosine kinase domain.

Human ProNGF: biological effects and binding profiles at TrkA, P75NTR and sortilin.

Nerve growth factor (NGF) promotes cell survival via binding to the tyrosine kinase receptor A (TrkA). Its precursor, proNGF, binds to p75(NTR) and sortilin receptors to initiate apoptosis. Current disagreement exists over whether proNGF acts neurotrophically following binding to TrkA. As in Alzheimer's disease the levels of proNGF increase and TrkA decrease, it is important to clarify the properties of proNGF. Here, wild-type and cleavage-resistant mutated forms (M) of proNGF were engineered and their binding characteristics determined. M-proNGF and NGF bound to p75(NTR) with similar affinities, whilst M-proNGF had a lower affinity than NGF for TrkA. M-proNGF behaved neurotrophically, albeit less effectively than NGF. M-proNGF addition resulted in phosphorylation of TrkA and ERK1/2, and in PC12 cells elicited neurite outgrowth and supported cell survival. Conversely, M-proNGF addition to cultured cortical neurons initiated caspase 3 cleavage. Importantly, these biological effects were shown to be mediated by unprocessed M-proNGF. Surprisingly, binding of the pro region alone to TrkA, at a site other than that of NGF, caused TrkA and ERK1/2 phosphorylation. Our data show that M-proNGF stimulates TrkA to a lesser degree than NGF, suggesting that in Alzheimer brain the increased proNGF : NGF and p75(NTR) : TrkA ratios may permit apoptotic effects to predominate over neurotrophic effects.

TrkAd5: A novel therapeutic agent for treatment of inflammatory pain and asthma.

Elevated levels of nerve growth factor have been linked to the onset and persistence of many pain-related disorders and asthma. Described here are the design, expression, refolding, and purification of a monomeric (nonstrand-swapped) form of the binding domain of the nerve growth factor receptor, designated TrkAd5. We have shown that TrkAd5 produced recombinantly binds nerve growth factor with picomolar affinity. TrkAd5 has been characterized using a variety of biophysical and biochemical assays and is shown here to be stable in both plasma and urine. The palliative effects of TrkAd5 are demonstrated in animal models of inflammatory pain and allergic asthma. We conclude that TrkAd5 will prove effective in ameliorating both acute and chronic conditions where nerve growth factor acts as a mediator and suggest a role for its application in vivo as a novel therapeutic.