Biphasic regulation of inositol trisphosphate (IP3)-stimulated Ca2+ mobilization by cytosolic Ca2+ is believed to contribute to regenerative intracellular Ca2+ signals. Since cells typically express several IP3 receptor isoforms and the effects of cytosolic Ca2+ are not mediated by a single mechanism, it is important to resolve the properties of each receptor subtype. Full-length rat types-1 and -3 IP3 receptors were expressed in insect Sf9 cells at levels 10-40-fold higher than the endogenous receptors. The expressed receptors were glycosylated and assembled into tetramers, and binding of [3H]IP3 to each subtype was regulated by cytosolic Ca2+. The effects of increased [Ca2+] on native cerebellar and type-1 receptors expressed in Sf9 cells were indistinguishable. A maximally effective increase in [Ca2+] reversibly inhibited [3H]IP3 binding by approx. 50% by decreasing the number of IP3-binding sites (Bmax) without affecting their affinity for IP3. The effects of Ca2+ on type-3 receptors were more complex: increasing [Ca2+] first stimulated [3H]IP3 binding by increasing Bmax, and then inhibited it by causing a substantial decrease in the affinity of the receptor for IP3. The different effects of Ca2+ on the receptor subtypes were not a consequence of limitations in the availability of accessory proteins or of artifactual effects of Ca2+ on membrane structure. We conclude that Ca2+ can inhibit IP3 binding to types-1 and -3 IP3 receptors although by different mechanisms, and that IP3 binding to type-3 receptors is stimulated at intermediate [Ca2+]. A consequence of these differences is that, at resting cytosolic [Ca2+], type-3 receptors are more sensitive than type-1 receptors to IP3, but the situation reverses at higher cytosolic [Ca2+]. Such differences may be important in generating the spatially and temporally complex changes in cytosolic [Ca2+] evoked by receptors linked to IP3 formation.

Calmodulin inhibits both inositol 1,4,5-trisphosphate (IP3) binding to, and IP3-evoked Ca2+ release by, cerebellar IP3 receptors [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U. S.A. 94, 11627-11632]. In the present study, full-length rat type-1 and -3 IP3 receptors were expressed at high levels in insect Spodoptera frugiperda 9 cells and the effects of calmodulin were examined. In the absence of Ca2+, calmodulin caused a concentration-dependent and reversible inhibition of [3H]IP3 binding to type-1 IP3 receptors by decreasing their apparent affinity for IP3. The effect was not reproduced by high concentrations of troponin C, parvalbumin or S-100. Increasing the medium free [Ca2+] ([Ca2+]m) inhibited [3H]IP3 binding to type-1 receptors, but the further inhibition caused by a submaximal concentration of calmodulin was similar at each [Ca2+]m. In the absence of Ca2+, 125I-calmodulin bound to a single site on each type-1 receptor subunit and to an additional site in the presence of Ca2+. There was no detectable binding of 125I-calmodulin to type-3 receptors and binding of [3H]IP3 was insensitive to calmodulin at all [Ca2+]m. Both peptide and conventional Ca2+-calmodulin antagonists affected neither [3H]IP3 binding directly nor the inhibitory effect of calmodulin in the absence of Ca2+, but each caused a [Ca2+]m-dependent reversal of the inhibition of [3H]IP3 binding caused by calmodulin. Camstatin, a peptide that binds to calmodulin equally well in the presence or absence of Ca2+, reversed the inhibitory effects of calmodulin on [3H]IP3 binding at all [Ca2+]m. We conclude that calmodulin specifically inhibits [3H]IP3 binding to type-1 IP3 receptors: the first example of a protein regulated by calmodulin in an entirely Ca2+-independent manner. Inhibition of type-1 IP3 receptors by calmodulin may dynamically regulate their sensitivity to IP3 in response to the changes in cytosolic free calmodulin concentration thought to accompany stimulation of neurones.

Spinal disease in dogs is commonly encountered in veterinary practice. Numerous diseases may cause similar clinical signs and presenting histories. The study objective was to use statistical models to identify combinations of discrete parameters from the patient signalment, history and neurological examination that could suggest the most likely diagnoses with statistical significance.

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There are three subtypes of mammalian Ins(1,4,5)P(3) (InsP(3)) receptor, each of which forms an intracellular Ca(2+) channel. Biphasic regulation of InsP(3) receptors by cytosolic Ca(2+) is well documented in cells expressing predominantly type 1 or type 2 InsP(3) receptors and might contribute to the regenerative recruitment of Ca(2+) release events and to limiting their duration in intact cells. The properties of type 3 receptors are less clear. Bilayer recording from InsP(3) receptors of RIN-5F cells, cells in which the InsP(3) receptors are likely to be largely type 3, recently suggested that the receptors are not inhibited by Ca(2+) [Hagar, Burgstahler, Nathanson and Ehrlich (1998) Nature (London) 296, 81-84]. By using antipeptide antisera that either selectively recognized each InsP(3) receptor subtype or interacted equally well with all subtypes, together with membranes from Spodoptera frugiperda (Sf9) cells expressing only single receptor subtypes to calibrate the immunoblotting, we quantified the relative levels of expression of type 1 (17%) and type 3 (77%) InsP(3) receptors in RINm5F cells. In unidirectional (45)Ca(2+) efflux experiments from permeabilized RINm5F cells, submaximal concentrations of InsP(3) released only a fraction of the InsP(3)-sensitive Ca(2+) stores, indicating that responses to InsP(3) are quantal. Increasing the cytosolic free [Ca(2+)] ([Ca(2+)](i)) from approx. 4 to 186 nM increased the sensitivity of the Ca(2+) stores to InsP(3): the EC(50) decreased from 281+/-15 to 82+/-2 nM. Further increases in [Ca(2+)](i) massively decreased the sensitivity of the stores to InsP(3), by almost 10-fold when [Ca(2+)](i) was 2.4 microM, and by more than 3000-fold when it was 100 microM. The inhibition caused by 100 microM Ca(2+) was fully reversed within 60 s of the restoration of [Ca(2+)](i) to 186 nM. The effect of submaximal InsP(3) concentrations on Ca(2+) mobilization from permeabilized RINm5F cells is therefore biphasically regulated by cytosolic Ca(2+). We conclude that type 3 InsP(3) receptors of RINm5F cells mediate quantal Ca(2+) release and they are biphasically regulated by cytosolic Ca(2+), either because a single type 1 subunit within the tetrameric receptor confers the Ca(2+) inhibition or because the type 3 subtype is itself directly inhibited by Ca(2+).

Objectives The aim of this study was to evaluate if a combination of discrete clinical characteristics can be used to identify the most likely differential diagnoses in cats with spinal disease. Methods Two hundred and twenty-one cats referred for further evaluation of spinal disease were included and categorised as follows: non-lymphoid neoplasia (n = 44); intervertebral disc disease (n = 42); fracture/luxation (n = 34); ischaemic myelopathy (n = 22); feline infectious peritonitis virus myelitis (n = 18); lymphoma (n = 16); thoracic vertebral canal stenosis (n = 11); acute non-compressive nucleus pulposus extrusion (n = 11); traumatic spinal cord contusion (n = 8); spinal arachnoid diverticula (n = 7); lumbosacral stenosis (n = 5); and spinal empyema (n = 3). Information retrieved from the medical records included signalment, clinical history and clinical presentation. Univariate analyses of variables (clinical history, breed, age, sex, general physical examination findings, onset, progression, spinal hyperaesthesia, asymmetry, ambulatory status and neuroanatomical location) were performed, and variables were retained in a multivariate logistic regression model if P <0.05. Results Multivariate logistic regression revealed that intervertebral disc disease most often occurred in middle-aged, purebred cats with a normal general physical examination and an acute onset of painful and progressive clinical signs. Ischaemic myelopathy occurred most often in older cats with a stable or improving, non-painful, lateralising, C6–T2 myelopathy. Spinal fracture/luxation occurred most often in younger cats and resulted most often in a peracute onset, painful, non-ambulatory neurological status. Concurrent systemic abnormalities or abnormal findings detected on general physical examination were significantly associated with feline infectious peritonitis virus myelitis, spinal lymphoma or spinal empyema. Conclusions and relevance This study suggests that using easily identifiable characteristics from the history and clinical examination can assist in obtaining a preliminary differential diagnosis when evaluating cats with spinal disease. This information could aid veterinary practitioners in clinical decision-making.

OBJECTIVE To assess the anatomic distribution of thoracolumbar and lumbar intervertebral disk extrusions (IVDEs) in English Cocker Spaniels as compared with findings in Dachshunds and to characterize clinical findings in English Cocker Spaniels with thoracolumbar or lumbar IVDEs affecting various regions of the vertebral column. DESIGN Retrospective observational study. ANIMALS 81 English Cocker Spaniels and 81 Dachshunds with IVDEs. PROCEDURES Signalment, clinical signs, neurologic examination findings, and affected intervertebral disk spaces (IVDSs) were recorded for both breeds. Management methods and outcomes were recorded for English Cocker Spaniels. Lesions were categorized as thoracolumbar (IVDSs T9–10 through L1–2), midlumbar (L2–3 through L4–5), or caudal lumbar (L5–6 through L7–S1). RESULTS Midlumbar and caudal lumbar IVDEs were significantly more common in English Cocker Spaniels than in Dachshunds. English Cocker Spaniels with caudal lumbar IVDEs had a longer median duration of clinical signs before evaluation and more commonly had unilateral pelvic limb lameness or spinal hyperesthesia as the predominant clinical sign than did those with IVDEs at other sites. Those with caudal lumbar IVDEs less commonly had neurologic deficits and had a higher median neurologic grade (indicating lesser severity), shorter mean postoperative hospitalization time, and faster mean time to ambulation after surgery than those with other sites affected. These variables did not differ between English Cocker Spaniels with thoracolumbar and midlumbar IVDEs. CONCLUSIONS AND CLINICAL RELEVANCE Caudal and midlumbar IVDEs were more common in English Cocker Spaniels than in Dachshunds. English Cocker Spaniels with caudal lumbar IVDE had clinical signs and posttreatment responses that differed from those in dogs with midlumbar or thoracolumbar IVDE.

Objectives To describe the clinical presentation, MRI findings and outcome in dogs with eosinophilic meningoencephalitis of unknown origin. Materials and Methods Dogs were included in this retrospective study if they had complete medical records, complete neurological examination, MR imaging, cerebellomedullary cerebrospinal fluid sample consistent with eosinophilic pleocytosis and negative infectious disease testing. Results Eleven dogs were included with a median age of 22·0 months (range 7·6 to 92·0 months). Nine breeds were represented. Neurological abnormalities included obtundation (n=10), menace response deficits (n=9), proprioceptive deficits (n=7), ataxia (n=7) and seizures (n=2). Neuroanatomical localisation was multi‐focal (n=4), central vestibular system (n=4), diffuse forebrain (n=2) or left trigeminal/facial nerves (n=1). Seven dogs had peripheral eosinophilia. Ten dogs had bilateral symmetrical lesions affecting the cortical grey matter, which was hyperintense on T2‐weighted and fluid‐attenuating inversion recovery images and iso‐ to hypointense on T1‐weighted images with associated meningeal contrast enhancement. MRI findings were consistent with diffuse meningitis and atrophy or necrosis of cortical grey matter. One dog had increased contrast uptake in the left trigeminal nerve. Ten dogs receiving corticosteroids survived to discharge, with seven also receiving cytarabine arabinoside. Median survival time was 762 days. Clinical Significance Eosinophilic meningoencephalitis of unknown origin affects younger larger‐breed dogs, with the majority having suspected diffuse cerebrocortical meningitis and cortical (polio)encephalitis, which can be identified on MRI. Response to immunosuppressive treatment is good in the medium to long term, although further studies are required in this area.