Sub-acute toxicity evaluation of aqueous leaf extract from Passiflora edulis Sims f. edulis (Gulupa) in Wistar rats.

The genus Passiflora (Passifloraceae) comprises about 500 species. The Passiflora edulis stands out because of its economic and medicinal importance. It is widely planted in tropical and subtropical regions worldwide, especially in South America, the Caribbean, South Africa, and Asia. The aqueous extract of Passiflora edulis Sims f. edulis (Gulupa) leaves is used in traditional medicine for its soothing and tranquilizing effects on the central nervous system. Therefore, evaluating its safety for human use is a fundamental requirement to continue the development of new therapies within the framework of regulatory, preclinical, and clinical guidelines. Here, the sub-acute toxicity study was conducted following the Organization for Economic Cooperation and Development (OECD) guideline 407 for 28 days in Wistar albino rats. The study showed that 1000 mg/kg/day of the aqueous extract in 10 adult Wistar rats (five males and five females) was well tolerated. The hematological results are at normal levels. However, monocytopenia and eosinopenia were observed with a significant difference (P < 0,05) for both male and female rats treated with the aqueous extract of Passiflora edulis. The results show that liver and kidney function profiles were conserved. However, an increase in ALT is observed with significant differences between male and female rats treated with the extract compared to the controls. Study findings were limited to non-adverse histopathological results of a slightly increased incidence of focal periportal lymphocytic infiltrate in the liver and focal corticomedullary nephrocalcinosis in the kidney compared to control. Therefore, the aqueous extract of Passiflora edulis has a good safety profile in oral administration, was well tolerated, and did not cause any lethality or adverse effects in the sub-acute toxicity study in male and female rats. The NOAEL (no observed adverse effect level) for the 28-day subacute toxicity study was considered to be 1000 mg/kg.

Dendritogenic Potential of the Ethanol Extract from Lippia alba Leaves in Rat Cortical Neurons.

A reduced dendritic complexity, especially in regions such as the hippocampus and the prefrontal cortex, has been linked to the pathophysiology of some neuropsychiatric disorders, in which synaptic plasticity and functions such as emotional and cognitive processing are compromised. For this reason, the identification of new therapeutic strategies would be enriched by the search for metabolites that promote structural plasticity. The present study evaluated the dendritogenic potential of the ethanol extract of Lippia alba, an aromatic plant rich in flavonoids and terpenes, which has been widely used in traditional medicine for its presumed analgesic, anxiolytic, and antidepressant potential. An in vitro model of rat cortical neurons was used to determine the kinetics of the plant's effect at different time intervals. Changes in morphological parameters of the neurons were determined, as well as the dendritic complexity, by Sholl analysis. The extract promotes the outgrowth of dendritic branching in a rapid and sustained fashion, without being cytotoxic to the cells. We found that this effect could be mediated by the phosphatidylinositol 3-kinase pathway, which is involved in mechanisms of neuronal plasticity, differentiation, and survival. The evidence presented in this study provides a basis for further research that, through in vivo models, can delve into the plant's therapeutic potential.

An update on neurobiological mechanisms involved in the development of chemotherapy-induced cognitive impairment (CICI).

Cancer is the second leading cause of death worldwide despite efforts in early diagnosis of the disease and advances in treatment. The use of drugs that exert toxic effects on tumor cells or chemotherapy is one of the most widely used treatments against cancer. However, its low toxic selectivity affects both healthy cells and cancer cells. It has been reported that chemotherapeutic drugs may generate neurotoxicity that induces deleterious effects of chemotherapy in the central nervous system. In this sense, patients report decreased cognitive abilities, such as memory, learning, and some executive functions after chemotherapy. This chemotherapy-induced cognitive impairment (CICI) develops during treatment and persists even after chemotherapy. Here we present a review of the literature on the main neurobiological mechanisms involved in CICI using a Boolean formula following the steps of the PRISMA guidelines that were used to perform statements searches in various databases. The main mechanisms described in the literature to explain CRCI include direct and indirect mechanisms that induce neurotoxicity by chemotherapeutic agents. Therefore, this review provides a general understanding of the neurobiological mechanisms of CICI and the possible therapeutic targets to prevent it..

Therapeutic potential of TRPM8 channels in cancer treatment.

Cancer is a multifactorial process associated with changes in signaling pathways leading to cell cycle variations and gene expression. The transient receptor potential melastatin 8 (TRPM8) channel is a non-selective cation channel expressed in neuronal and non-neuronal tissues, where it is involved in several processes, including thermosensation, differentiation, and migration. Cancer is a multifactorial process associated with changes in signaling pathways leading to variations in cell cycle and gene expression. Interestingly, it has been shown that TRPM8 channels also participate in physiological processes related to cancer, such as proliferation, survival, and invasion. For instance, TRPM8 channels have an important role in the diagnosis, prognosis, and treatment of prostate cancer. In addition, it has been reported that TRPM8 channels are involved in the progress of pancreatic, breast, bladder, colon, gastric, and skin cancers, glioblastoma, and neuroblastoma. In this review, we summarize the current knowledge on the role of TRPM8 channels in cancer progression. We also discuss the therapeutic potential of TRPM8 in carcinogenesis, which has been proposed as a molecular target for cancer therapy.

TRP Channels as Molecular Targets to Relieve Cancer Pain.

Transient receptor potential (TRP) channels are critical receptors in the transduction of nociceptive stimuli. The microenvironment of diverse types of cancer releases substances, including growth factors, neurotransmitters, and inflammatory mediators, which modulate the activity of TRPs through the regulation of intracellular signaling pathways. The modulation of TRP channels is associated with the peripheral sensitization observed in patients with cancer, which results in mild noxious sensory stimuli being perceived as hyperalgesia and allodynia. Secondary metabolites derived from plant extracts can induce the activation, blocking, and desensitization of TRP channels. Thus, these compounds could act as potential therapeutic agents, as their antinociceptive properties could be beneficial in relieving cancer-derived pain. In this review, we will summarize the role of TRPV1 and TRPA1 in pain associated with cancer and discuss molecules that have been reported to modulate these channels, focusing particularly on the mechanisms of channel activation associated with molecules released in the tumor microenvironment.

TRP Channels Role in Pain Associated With Neurodegenerative Diseases.

Transient receptor potential (TRP) are cation channels expressed in both non-excitable and excitable cells from diverse tissues, including heart, lung, and brain. The TRP channel family includes 28 isoforms activated by physical and chemical stimuli, such as temperature, pH, osmotic pressure, and noxious stimuli. Recently, it has been shown that TRP channels are also directly or indirectly activated by reactive oxygen species. Oxidative stress plays an essential role in neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases, and TRP channels are involved in the progression of those diseases by mechanisms involving changes in the crosstalk between Ca2+ regulation, oxidative stress, and production of inflammatory mediators. TRP channels involved in nociception include members of the TRPV, TRPM, TRPA, and TRPC subfamilies that transduce physical and chemical noxious stimuli. It has also been reported that pain is a complex issue in patients with Alzheimer's and Parkinson's diseases, and adequate management of pain in those conditions is still in discussion. TRPV1 has a role in neuroinflammation, a critical mechanism involved in neurodegeneration. Therefore, some studies have considered TRPV1 as a target for both pain treatment and neurodegenerative disorders. Thus, this review aimed to describe the TRP-dependent mechanism that can mediate pain sensation in neurodegenerative diseases and the therapeutic approach available to palliate pain and neurodegenerative symptoms throughout the regulation of these channels.

Role of Connexins 30, 36, and 43 in Brain Tumors, Neurodegenerative Diseases, and Neuroprotection.

Gap junction (GJ) channels and their connexins (Cxs) are complex proteins that have essential functions in cell communication processes in the central nervous system (CNS). Neurons, astrocytes, oligodendrocytes, and microglial cells express an extraordinary repertory of Cxs that are important for cell to cell communication and diffusion of metabolites, ions, neurotransmitters, and gliotransmitters. GJs and Cxs not only contribute to the normal function of the CNS but also the pathological progress of several diseases, such as cancer and neurodegenerative diseases. Besides, they have important roles in mediating neuroprotection by internal or external molecules. However, regulation of Cx expression by epigenetic mechanisms has not been fully elucidated. In this review, we provide an overview of the known mechanisms that regulate the expression of the most abundant Cxs in the central nervous system, Cx30, Cx36, and Cx43, and their role in brain cancer, CNS disorders, and neuroprotection. Initially, we focus on describing the Cx gene structure and how this is regulated by epigenetic mechanisms. Then, the posttranslational modifications that mediate the activity and stability of Cxs are reviewed. Finally, the role of GJs and Cxs in glioblastoma, Alzheimer's, Parkinson's, and Huntington's diseases, and neuroprotection are analyzed with the aim of shedding light in the possibility of using Cx regulators as potential therapeutic molecules.

Activation of BK Channel Contributes to PL-Induced Mesenchymal Stem Cell Migration.

Due to their capacity to proliferate, migrate, and differentiate, mesenchymal stem cells (MSCs) are considered to be good candidates for regenerative medicine applications. The mechanisms underlying proliferation and differentiation of MSCs have been studied. However, much less is known about the mechanisms regulating the migration of MSCs. Platelet lysate (PL), a supplement used to promote cell expansion, has been shown to promote MSCs migration; however, the underlying mechanism are unknown. Here, by using adipose-derived rat MSCs (rMSCs) and the scratch assay in the absence and presence of various BK channels modulators, we evaluated the role of BK channels in mediating the PL-stimulated migration of rMSCs. We found that 5% PL increased rMSCs migration, and this effect was blocked by the addition of the BK channel selective antagonist Iberiotoxin (IBTX). In the absence of PL, the BK channel agonist NS1619, stimulated rMSCs migration to similar level as 5% PL. Addition of both NS1619 and 5% PL resulted in an increase in rMSCs migration, that was higher than when either one was added individually. From whole-cell recordings, it was found that the addition of 5% PL increased the magnitude of BK current density. By using Western blot and flow cytometry, it was found that PL did not affect the expression of BK channels. Together, our results indicate that as shown in other cell types, activation of BK channels by themselves also promote rMSC migration, and show that activation of BK channels contribute to the observed PL-induced increase in migration of rMSC.

Connexin-Mediated Functional and Metabolic Coupling Between Astrocytes and Neurons.

The central nervous system (CNS) requires sophisticated regulation of neuronal activity. This modulation is partly accomplished by non-neuronal cells, characterized by the presence of transmembrane gap junctions (GJs) and hemichannels (HCs). This allows small molecule diffusion to guarantee neuronal synaptic activity and plasticity. Astrocytes are metabolically and functionally coupled to neurons by the uptake, binding and recycling of neurotransmitters. In addition, astrocytes release metabolites, such as glutamate, glutamine, D-serine, adenosine triphosphate (ATP) and lactate, regulating synaptic activity and plasticity by pre- and postsynaptic mechanisms. Uncoupling neuroglial communication leads to alterations in synaptic transmission that can be detrimental to neuronal circuit function and behavior. Therefore, understanding the pathways and mechanisms involved in this intercellular communication is fundamental for the search of new targets that can be used for several neurological disease treatments. This review will focus on molecular mechanisms mediating physiological and pathological coupling between astrocytes and neurons through GJs and HCs.

Cytoprotective action against oxidative stress in astrocytes and neurons by Bactris guineensis (L.) H.E. Moore (corozo) fruit extracts.

Neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis are characterized by having a significant increase in oxidative stress that can lead to the cellular damage of both neurons and astrocytes. Therefore, the search for molecules that can modulate oxidative stress in these diseases has recently gain interest, especially for those non-traditional antioxidants that can be gained from diet. In the present work, pulp and seed extracts from the fruit of the palm, Bactris guineensis were obtained by hydro-alcoholic solution and by a solid-liquid phase using solvents with different polarities and evaluated for their capacity to protect both neurons and astrocytes against rotenone-induced oxidative stress. Analysis of the chemical antioxidant activity showed that Bactris guineensis pulp crude extract and seed ethyl acetate and ethanol extracts had a high scavenging capacity when compared with extracts obtained in Hexane and dichloromethane. Toxicity assays also showed that the pulp crude extract and seed ethyl acetate and ethanol extracts at low doses did not affect the cell viability of primary astrocyte and SH-SY5Y neuroblastoma cells. In addition, ethyl acetate and ethanol extracts, not only decreased O2- radicals production but also protected both SHSY5Y and astrocytes oxidative stress induced by rotenone. Together our results suggest that Bactris guineensis fruit contain antioxidant molecules that can have therapeutic potential.

Cellular and molecular mechanisms of antioxidants in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative movement disorder characterized by the degeneration and progressive loss of dopaminergic neurons in the substantia nigra pars compacta. It has been suggested that oxidative stress plays a role in the etiology and progression of PD. For instance, low levels of endogenous antioxidants, increased reactive species, augmented dopamine oxidation, and high iron levels have been found in brains from PD patients. In vitro and in vivo studies of Parkinson models evaluating natural and endogenous antioxidants such as polyphenols, coenzyme Q10, and vitamins A, C, and E have shown protective effects against oxidative-induced neuronal death. In this paper, we will review the mechanisms by which polyphenols and endogenous antioxidants can produce protection. Some of the mechanisms reviewed include: scavenging nitrogen and oxygen reactive species, regulation of signaling pathways associated with cell survival and inflammation, and inhibition of synphilin-1 and alpha-synuclein aggregation.

Preparation of adult spinal cord motor neuron cultures under serum-free conditions.

Spinal cord motor neuron cultures are an important tool for the study of mechanisms involved in motor neuron survival, degeneration and regeneration, volatile anesthetic-induced immobility, motor neuron disorders such as amyotrophic lateral sclerosis or spinal muscular atrophy as well as in spinal cord injury. Embryonic spinal cord motor neurons derived from rats have been successfully cultured; unfortunately, the culture of adult motor neurons has been problematic due to their short-term survival. Recently, by using a cocktail of target-derived factors, neurotrophins (brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor) and a permeable cyclic adenosine monophosphate analog, we have established a reproducible protocol for long-term cultures of healthy and functional adult motor neurons (Exp Neurol 220:303-315, 2009). Here, we now describe in detail the steps that we used for the optimization of the process of isolation and maintenance of adult rat ventral horn motor neurons in vitro.

Effects of natural antioxidants in neurodegenerative disease.

Polyphenols are secondary metabolites with antioxidant properties and are abundant in the diet. Fruits, vegetables, herbs, and various drinks (tea, wine, and juices) are all sources of these molecules. Despite their abundance, investigations into the benefits of polyphenols in human health have only recently begun. Phenolic compounds have received increasing interest because of numerous epidemiological studies. These studies have suggested associations between the consumption of polyphenol-rich aliments and the prevention of chronic diseases, such as cancer, cardiovascular diseases, and neurodegenerative diseases. More specifically, in the last 10 years literature on the neuroprotective effects of a polyphenol-rich diet has grown considerably. It has been demonstrated, in various cell culture and animal models, that these metabolites are able to protect neuronal cells by attenuating oxidative stress and damage. However, it remains unclear as to how these compounds reach the brain, what concentrations are necessary, and what biologically active forms are needed to exert beneficial effects. Therefore, further research is needed to identify the molecular pathways and intracellular targets responsible for polyphenol's neuroprotective effects. The aim of this paper is to present various well-known dietary polyphenols and their mechanisms of neuroprotection with an emphasis on Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.

Regulation of inhibitory neurotransmission by the scaffolding protein ankyrin repeat-rich membrane spanning/kinase D-interacting substrate of 220 kDa.

Scaffolding proteins play a critical role in the proper development and function of neural circuits. In contrast to the case for excitatory circuits, in which the role of several scaffolding proteins has been characterized, less is known about the scaffolding proteins that regulate inhibitory neurotransmission. The ankyrin repeat-rich membrane spanning (ARMS)/kinase D-interacting substrate of 220 kDa (Kidins220) scaffolding protein is expressed during the establishment of γ-aminobutyric acid (GABA) neurotransmission and is highly regulated by activity. To evaluate whether ARMS/Kidins220 expression affects GABAergic neurotransmission, we modified the ARMS/Kidins220 levels during the period of its maximum expression in culture (DIV 1-10). Whereas a decrease in ARMS/Kidins220 levels suppressed GABAergic neurotransmission, overexpression of ARMS/Kidins220 produced an increase in GABAergic neurotransmission in hippocampal neurons. In addition, we found that ARMS/Kidins220 regulates GABAergic neurotransmission by a presynaptic mechanism. Our results suggest that the ARMS/Kidins220 scaffold protein plays a critical role in the regulation of inhibitory transmission in hippocampal neurons.

Isoflurane inhibits cyclic adenosine monophosphate response element-binding protein phosphorylation and calmodulin translocation to the nucleus of SH-SY5Y cells.

BACKGROUND: Calmodulin (CaM) activation by Ca(2+), its translocation to the nucleus, and stimulation of phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) (P-CREB) are necessary for new gene expression and have been linked to long-term potentiation, a process important in memory formation. Because isoflurane affects memory, we tested whether isoflurane interfered with the translocation of CaM to the neuronal cell nucleus and attenuated the formation P-CREB. METHODS: SH-SY5Y cells, a human neuroblastoma cell line, were cultured. Cells were depolarized with KCl and the phosphorylation of CREB examined by Western blotting, enzyme-linked immunosorbant assay, and immunocytochemistry. The translocation of CaM from the cytosol to the nucleus was also examined after depolarization. Cells were depolarized and lysed and fractionated by centrifugation to determine the amount of CaM translocated to the nucleus. CaM was localized by immunocytochemistry and quantitated by Western blotting and imaging. Before and during KCl depolarization, cells were exposed to isoflurane, isoflurane plus Bay K 8644, nitrendipine, and omega-conotoxin GVIa, respectively. RESULTS: P-CREB increased after KCl depolarization. The increase of P-CREB peaked at depolarization duration of 30 s. The increase in P-CREB formation was inhibited by nitrendipine, but not omega-conotoxin, and by isoflurane in a concentration-dependent fashion. Pretreatment with the L-type Ca(2+) channel agonist, Bay K 8644, attenuated the inhibition of P-CREB formation by isoflurane. CaM presence in the nucleus occurred after KCl depolarization. CaM translocation was inhibited by nitrendipine and attenuated by isoflurane. Bay K 8644 pretreatment decreased the isoflurane inhibition of CaM translocation to the nucleus. CONCLUSIONS: Our data demonstrate that isoflurane inhibits CaM translocation and P-CREB formation. This most likely occurs through isoflurane inhibition of Ca(2+)entry through L-type Ca(2+) channels.

Muscle-conditioned media and cAMP promote survival and neurite outgrowth of adult spinal cord motor neurons.

Embryonic spinal cord motor neurons (MNs) can be maintained in vitro for weeks with a cocktail of trophic factors and muscle-derived factors under serum-containing conditions. Here we investigated the beneficial effects of muscle-derived factors in the form of muscle-conditioned medium (MCM) on the survival and neurite outgrowth of adult rat spinal cord MNs under serum-free conditions. Ventral horn dissociated cell cultures from the cervical enlargement were maintained in the presence of one or more of the following factors: brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), a cell permeant cyclic adenosine-3',5'-monophosphate (cAMP) analog and MCM. The cell cultures were immunostained with several antibodies recognizing a general neuronal marker the microtubule-associated protein 2 (MAP2) and either one or more motor neuronal markers: the non-phosphorylated neurofilament heavy isoform (SMI32), the transcription factors HB9 and Islet-1 and the choline acetyl transferase. We found that treatment with MCM together with the cAMP analog was sufficient to promote selective survival and neurite outgrowth of adult spinal cord MNs. These conditions can be used to maintain adult spinal cord MNs in dissociated cultures for several weeks and may have therapeutic potential following spinal cord injury or motor neuropathies. More studies are necessary to evaluate how MCM and the cAMP analog act in synergy to promote the survival and neurite outgrowth of adult MNs.

Pulses of extracellular K+ produce two cytosolic Ca2+ transients that display different temperature dependence and carbonyl cyanide m-chlorophenyl sensitivity in SH-SY5Y cells.

In SH-SY5Y cells we have shown that stimulation with high extracellular K+ ([K+]e) evokes a transient increase in cytoplasmic Ca2+ ([Ca2+]cyt) (K+on) that is triggered by the opening of voltage-dependent Ca2+ channels and followed by Ca2+ -induced Ca2+ release from the endoplasmic reticulum (Xu, F., Zhang, J., Recio-Pinto, E. and Blanck, T.J., Halothane and isoflurane augment depolarization-induced cytosolic CA2+ transients and attenuate carbachol-stimulated CA2+ transients, Anesthesiology, 92 (2000) 1746-56). The removal of high-[K+]e results in a second transient increase in [Ca2+]cyt (K+off) that is independent of extracellular Ca2+ (Corrales, A., Montoya, G.J., Sutachan, J.J., Cornillez-Ty, G., Garavito-Aguilar, Z., Xu, F., Blanck, T.J. and Recio-Pinto, E., Transient increases in extracellular K+ produce two pharmacological distinct cytosolic Ca2+ transients, Brain Res., 1031 (2005) 174-184). In this study we further characterize the properties of K+off. We found that K+off was detectable at near physiological temperatures (34-36 degrees C) but, depending on the level of [K+]e, it was undetectable or highly diminished at room temperature. In contrast, K+on was increased by lowering the temperature. Extracellular Na+ -replacement with K+ did not affect K+off, indicating that K+off was not generated by osmolarity changes. Replacement of extracellular Na+ with choline+ did not affect K+off, indicating that K+off did not result from activity changes of the plasma membrane Na+/Ca2+ exchanger. Caffeine decreased K+on but not K+off. CCCP (carbonyl cyanide m-chlorophenyl), a protonophore uncoupler that decreases mitochondrial Ca2+ uptake, decreased K+on but not K+off. CGP37157, an inhibitor of the mitochondria Na+/Ca2+ exchanger, decreased K+off when added alone but not when added simultaneously with CCCP. Clonazepam had similar effects as CGP37157. These findings indicate that the generation of K+off is strongly temperature-dependent and its pharmacology is distinct from the [Ca2+]cyt changes observed previously at room temperature.

Pluronic F-127 affects the regulation of cytoplasmic Ca2+ in neuronal cells.

Fura-2 is one of the most widely used cytoplasmic Ca2+ ([Ca2+]cyt) sensors. In studies using isolated dorsal root ganglion (DRG) neurons, the loading of Fura-2 AM is often facilitated by the use of pluronic F-127. In preliminary studies, we detected that the use of pluronic F-127 appeared to be affecting the depolarization-evoked [Ca2+]cyt transient in DRG neurons. To determine whether this was the case, we conducted a systematic study. Adult rat DRG neurons were cultured, and their response to 50 mM KCl was measured in sister cultured cells (isolated on the same day) that were loaded with 5 microM Fura-2AM in the absence or in the presence of 0.02% pluronic F-127. In the absence of pluronic F-127, the KCl-evoked [Ca2+]cyt transient changed with time, being wider on day 1 than on day 2 after plating. On day 2, the KCl-evoked [Ca2+]cyt transient was wider in neurons Fura-2 loaded in the presence of pluronic F-127. These results indicate that pluronic F-127 significantly alters depolarization-evoked [Ca2+]cyt transients, which may reflect alteration in regulation of [Ca2+]cyt in neuronal cells.

Transient increases in extracellular K+ produce two pharmacological distinct cytosolic Ca2+ transients.

Transient increases in extracellular K+ are observed under various conditions, including repetitive neuronal firing, anoxia, ischemia and hypoglycemic coma. We studied changes in cytoplasmic Ca2+ ([Ca2+]cyt) evoked by pulses of KCl in human neuroblastoma SH-SY5Y cells and rat dorsal root ganglia (DRG) neurons at 37 degrees C. A "pulse" of KCl evoked two transient increases in [Ca2+]cyt, one upon addition of KCl (K+on) and the other upon removal of KCl (K+off). The K+on transient has been described in many cell types and is initiated by the activation of voltage-dependent Ca2+ channels followed by Ca2+-evoked Ca2+ release from intracellular Ca2+ stores. The level of KCl necessary to evoke the K+off transient depends on the type of neuron, in SH-SY5Y cells it required 100 mM KCl, in most (but not all) of dorsal root ganglia neurons it could be detected with 100-200 mM KCl and in a very few dorsal root ganglia neurons it was detectable at 20-50 mM KCl. In SH-SY5Y cells, reduction of extracellular Ca2+ inhibited the K+on more strongly than the K+off and slowed the decay of K+off. Isoflurane (1 mM) reduced the K+on)- but not the K+off-peak. However, isoflurane slowed the decay of K+off. The nonspecific cationic channel blocker La3+ (100 microM) had an effect similar to that of isoflurane. Treatment with thapsigargin (TG) at a concentration known to only deplete IP3-sensitive Ca2+ stores did not affect K+on or K+off, suggesting that Ca2+ release from the IP3-sensitive Ca2+ stores does not contribute to K+on and K+off transients and that the thapsigargin-sensitive Ca2+ ATPases do not contribute significantly to the rise or decay rates of these transients. These findings indicate that a pulse of extracellular K+ produces two distinct transient increases in [Ca2+]cyt.