KATP channel activity and slow oscillations in pancreatic beta cells are regulated by mitochondrial ATP production.

Pancreatic beta cells secrete insulin in response to plasma glucose. The ATP-sensitive potassium channel (KATP ) links glucose metabolism to islet electrical activity in these cells by responding to increased cytosolic [ATP]/[ADP]. It was recently proposed that pyruvate kinase (PK) in close proximity to beta cell KATP locally produces the ATP that inhibits KATP activity. This proposal was largely based on the observation that applying phosphoenolpyruvate (PEP) and ADP to the cytoplasmic side of excised inside-out patches inhibited KATP . To test the relative contributions of local vs. mitochondrial ATP production, we recorded KATP activity using mouse beta cells and INS-1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of KATP activity by PEP + ADP. However, when the pH of the PEP solutions was not corrected for the addition of PEP, strong channel inhibition was observed as a result of the well-known action of protons to inhibit KATP . In cell-attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on KATP channel closure by glucose, further suggesting that PK is not an important regulator of KATP . In contrast, addition of mitochondrial inhibitors robustly increased KATP activity. Finally, by measuring the [ATP]/[ADP] responses to imposed calcium oscillations in mouse beta cells, we found that oxidative phosphorylation could raise [ATP]/[ADP] even when ADP was at its nadir during the burst silent phase, in agreement with our mathematical model. These results indicate that ATP produced by mitochondrial oxidative phosphorylation is the primary controller of KATP in pancreatic beta cells. KEY POINTS: Phosphoenolpyruvate (PEP) plus adenosine diphosphate does not inhibit KATP activity in excised patches. PEP solutions only inhibit KATP activity if the pH is unbalanced. Modulating pyruvate kinase has minimal effects on KATP activity. Mitochondrial inhibition, in contrast, robustly potentiates KATP activity in cell-attached patches. Although the ADP level falls during the silent phase of calcium oscillations, mitochondria can still produce enough ATP via oxidative phosphorylation to close KATP . Mitochondrial oxidative phosphorylation is therefore the main source of the ATP that inhibits the KATP activity of pancreatic beta cells.

The astroglial and stem cell functions of adult rat folliculostellate cells.

The mammalian pituitary gland is a complex organ consisting of hormone-producing cells, anterior lobe folliculostellate cells (FSCs), posterior lobe pituicytes, vascular pericytes and endothelial cells, and Sox2-expressing stem cells. We present single-cell RNA sequencing and immunohistofluorescence analyses of pituitary cells of adult female rats with a focus on the transcriptomic profiles of nonhormonal cell types. Samples obtained from whole pituitaries and separated anterior and posterior lobe cells contained all expected pituitary resident cell types and lobe-specific vascular cell subpopulations. FSCs and pituicytes expressed S100B, ALDOC, EAAT1, ALDH1A1, and VIM genes and proteins, as well as other astroglial marker genes, some common and some cell type-specific. We also found that the SOX2 gene and protein were expressed in ~15% of pituitary cells, including FSCs, pituicytes, and a fraction of hormone-producing cells, arguing against its stem cell specificity. FSCs comprised two Sox2-expressing subclusters; FS1 contained more cells but lower genetic diversity, while FS2 contained proliferative cells, shared genes with hormone-producing cells, and expressed genes consistent with stem cell niche formation, regulation of cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. FS1 cells were randomly distributed in the anterior and intermediate lobes, while FS2 cells were localized exclusively in the marginal zone between the anterior and intermediate lobes. These data indicate the identity of the FSCs as anterior pituitary-specific astroglia, with FS1 cells representing differentiated cells equipped for classical FSC roles and FS2 cells exhibiting additional stem cell-like features.

Ca2+ release or Ca2+ entry, that is the question: what governs Ca2+ oscillations in pancreatic ß cells?

The standard model for Ca2+ oscillations in insulin-secreting pancreatic ß cells centers on Ca2+ entry through voltage-activated Ca2+ channels. These work in combination with ATP-dependent K+ channels, which are the bridge between the metabolic state of the cells and plasma membrane potential. This partnership underlies the ability of the ß cells to secrete insulin appropriately on a minute-to-minute time scale to control whole body plasma glucose. Though this model, developed over more than 40 years through many cycles of experimentation and mathematical modeling, has been very successful, it has been challenged by a hypothesis that calcium-induced calcium release from the endoplasmic reticulum through ryanodine or inositol trisphosphate (IP3) receptors is instead the key driver of islet oscillations. We show here that the alternative model is in fact incompatible with a large body of established experimental data and that the new observations offered in support of it can be better explained by the standard model.

Oscillations in K(ATP) conductance drive slow calcium oscillations in pancreatic ß-cells.

ATP-sensitive K+ (K(ATP)) channels were first reported in the ß-cells of pancreatic islets in 1984, and it was soon established that they are the primary means by which the blood glucose level is transduced to cellular electrical activity and consequently insulin secretion. However, the role that the K(ATP) channels play in driving the bursting electrical activity of islet ß-cells, which drives pulsatile insulin secretion, remains unclear. One difficulty is that bursting is abolished when several different ion channel types are blocked pharmacologically or genetically, making it challenging to distinguish causation from correlation. Here, we demonstrate a means for determining whether activity-dependent oscillations in K(ATP) conductance play the primary role in driving electrical bursting in ß-cells. We use mathematical models to predict that if K(ATP) is the driver, then contrary to intuition, the mean, peak, and nadir levels of ATP/ADP should be invariant to changes in glucose within the concentration range that supports bursting. We test this in islets using Perceval-HR to image oscillations in ATP/ADP. We find that mean, peak, and nadir levels are indeed approximately invariant, supporting the hypothesis that oscillations in K(ATP) conductance are the main drivers of the slow bursting oscillations typically seen at stimulatory glucose levels in mouse islets. In conclusion, we provide, for the first time to our knowledge, causal evidence for the role of K(ATP) channels not only as the primary target for glucose regulation but also for their role in driving bursting electrical activity and pulsatile insulin secretion.

Cell-Type-Specific Expression Pattern of Proton-Sensing Receptors and Channels in Pituitary Gland.

In mammalian cells, extracellular protons act as orthosteric and allosteric ligands for multiple receptors and channels. The aim of this study is to identify proton sensors in the rat pituitary gland. qRT-PCR analysis indicated the expression of G-protein-coupled receptor 68 gene (Gpr68) and acid-sensing ion channel (ASIC) genes Asic1, Asic2, and Asic4 in anterior pituitary cells and Asic1 and Asic2 in immortalized GH3 pituitary cells. Asic1a and Asic2b were the dominant splice isoforms. Single anterior pituitary cell RNA sequencing and immunocytochemical analysis showed that nonexcitable folliculostellate cells express GPR68 gene and protein, whereas excitable secretory cells express ASIC genes and proteins. Asic1 was detected in all secretory cell types, Asic2 in gonadotrophs, thyrotrophs, and somatotrophs, and Asic4 in lactotrophs. Extracellular acidification activated two types of currents in a concentration-dependent manner: a fast-developing, desensitizing current with an estimated EC50-value of pH 6.7 and a slow-developing, non-desensitizing current that required a higher proton concentration for activation. The desensitizing current was abolished by removal of bath sodium and application of amiloride, a blocker of ASIC channels, whereas the non-desensitizing current was amiloride insensitive and voltage dependent. Activation of both currents increased the excitability of secretory pituitary cells, consistent with their potential physiological relevance in control of voltage-gated calcium influx and calcium-dependent cellular functions.

Modeling the diversity of spontaneous and agonist-induced electrical activity in anterior pituitary corticotrophs.

Pituitary corticotrophs fire action potentials spontaneously and in response to stimulation with corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), and such electrical activity is critical for calcium signaling and calcium-dependent adrenocorticotropic hormone secretion. These cells typically fire tall, sharp action potentials when spontaneously active, but a variety of other spontaneous patterns have also been reported, including various modes of bursting. There is variability in reports of the fraction of corticotrophs that are electrically active, as well as their patterns of activity, and the sources of this variation are not well understood. The ionic mechanisms responsible for CRH- and AVP-triggered electrical activity in corticotrophs are also poorly characterized. We use electrophysiological measurements and mathematical modeling to investigate possible sources of variability in patterns of spontaneous and agonist-induced corticotroph electrical activity. In the model, variation in as few as two parameters can give rise to many of the types of patterns observed in electrophysiological recordings of corticotrophs. We compare the known mechanisms for CRH, AVP, and glucocorticoid actions and find that different ionic mechanisms can contribute in different but complementary ways to generate the complex time courses of CRH and AVP responses. In summary, our modeling suggests that corticotrophs have several mechanisms at their disposal to achieve their primary function of pacemaking depolarization and increased electrical activity in response to CRH and AVP.NEW & NOTEWORTHY We and others recently demonstrated that the electrical activity and calcium dynamics of corticotrophs are strikingly diverse, both spontaneously and in response to the agonists CRH and AVP. Here we demonstrate this diversity with electrophysiological measurements and use mathematical modeling to investigate its possible sources. We compare the known mechanisms of agonist-induced activity in the model, showing how the context of ionic conductances dictates the effects of agonists even when their target is fixed.

Deconstructing the integrated oscillator model for pancreatic ß-cells.

Electrical bursting oscillations in the ß-cells of pancreatic islets have been a focus of investigation for more than fifty years. This has been aided by mathematical models, which are descendants of the pioneering Chay-Keizer model. This article describes the key biophysical and mathematical elements of this model, and then describes the path forward from there to the Integrated Oscillator Model (IOM). It is both a history and a deconstruction of the IOM that describes the various elements that have been added to the model over time, and the motivation for adding them. Finally, the article is a celebration of the 40th anniversary of the publication of the Chay-Keizer model.

Pituitary corticotroph identity and receptor-mediated signaling: a transcriptomics perspective.

Recent single-cell RNA sequencing has offered an unprecedented view of pituitary cell transcriptomic profiles. In this review, these new data are briefly discussed and compared with the classical literature, focusing on pituitary corticotrophs. These cells are introduced by discussing their marker genes, followed by a review of G protein-coupled receptor gene expression, heterotrimeric G protein genes, and genes encoding signaling pathways downstream of G proteins: adenylate cyclases, phosphodiesterases, phospholipases, and protein kinases. The expression patterns of enzyme-linked plasma membrane and nuclear hormone receptor genes was also analyzed. The overview of these selected groups of genes sheds new light on corticotrophic receptors and their signaling pathways and provides guidance for further basic and clinical research by identifying genes that not been studied so far.

Pituitary adenomas evade apoptosis via noxa deregulation in Cushing's disease.

Sporadic pituitary adenomas occur in over 10% of the population. Hormone-secreting adenomas, including those causing Cushing's disease (CD), cause severe morbidity and early mortality. Mechanistic studies of CD are hindered by a lack of in vitro models and control normal human pituitary glands. Here, we surgically annotate adenomas and adjacent normal glands in 25 of 34 patients. Using single-cell RNA sequencing (RNA-seq) analysis of 27594 cells, we identify CD adenoma transcriptomic signatures compared with adjacent normal cells, with validation by bulk RNA-seq, DNA methylation, qRT-PCR, and immunohistochemistry. CD adenoma cells include a subpopulation of proliferating, terminally differentiated corticotrophs. In CD adenomas, we find recurrent promoter hypomethylation and transcriptional upregulation of PMAIP1 (encoding pro-apoptotic BH3-only bcl-2 protein noxa) but paradoxical noxa downregulation. Using primary CD adenoma cell cultures and a corticotroph-enriched mouse cell line, we find that selective proteasomal inhibition with bortezomib stabilizes noxa and induces apoptosis, indicating its utility as an anti-tumor agent.

Symbiosis of Electrical and Metabolic Oscillations in Pancreatic ß-Cells.

Insulin is secreted in a pulsatile pattern, with important physiological ramifications. In pancreatic ß-cells, which are the cells that synthesize insulin, insulin exocytosis is elicited by pulses of elevated intracellular Ca2+ initiated by bursts of electrical activity. In parallel with these electrical and Ca2+ oscillations are oscillations in metabolism, and the periods of all of these oscillatory processes are similar. A key question that remains unresolved is whether the electrical oscillations are responsible for the metabolic oscillations via the effects of Ca2+, or whether the metabolic oscillations are responsible for the electrical oscillations due to the effects of ATP on ATP-sensitive ion channels? Mathematical modeling is a useful tool for addressing this and related questions as modeling can aid in the design of well-focused experiments that can test the predictions of particular models and subsequently be used to improve the models in an iterative fashion. In this article, we discuss a recent mathematical model, the Integrated Oscillator Model (IOM), that was the product of many years of development. We use the model to demonstrate that the relationship between calcium and metabolism in beta cells is symbiotic: in some contexts, the electrical oscillations drive the metabolic oscillations, while in other contexts it is the opposite. We provide new insights regarding these results and illustrate that what might at first appear to be contradictory data are actually compatible when viewed holistically with the IOM.

Calcium-Prolactin Secretion Coupling in Rat Pituitary Lactotrophs Is Controlled by PI4-Kinase Alpha.

The role of calcium, but not of other intracellular signaling molecules, in the release of pituitary hormones by exocytosis is well established. Here, we analyzed the contribution of phosphatidylinositol kinases (PIKs) to calcium-driven prolactin (PRL) release in pituitary lactotrophs: PI4Ks - which control PI4P production, PIP5Ks - which synthesize PI(4, 5)P2 by phosphorylating the D-5 position of the inositol ring of PI4P, and PI3KCs - which phosphorylate PI(4, 5)P2 to generate PI(3, 4, 5)P3. We used common and PIK-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs. Gene expression was analyzed by single-cell RNA sequencing and qRT-PCR analysis; intracellular and released hormones were assessed by radioimmunoassay and ELISA; and single-cell calcium signaling was recorded by Fura 2 imaging. Single-cell RNA sequencing revealed the expression of Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in lactotrophs. Wortmannin, a PI3K and PI4K inhibitor, but not LY294002, a PI3K inhibitor, blocked spontaneous action potential driven PRL release with a half-time of ~20 min when applied in 10 µM concentration, leading to accumulation of intracellular PRL content. Wortmannin also inhibited increase in PRL release by high potassium, the calcium channel agonist Bay K8644, and calcium mobilizing thyrotropin-releasing hormone without affecting accompanying calcium signaling. GSK-A1, a specific inhibitor of PI4KA, also inhibited calcium-driven PRL secretion without affecting calcium signaling and Prl expression. In contrast, PIK93, a specific inhibitor of PI4KB, and ISA2011B and UNC3230, specific inhibitors of PIP5K1A and PIP5K1C, respectively, did not affect PRL release. These experiments revealed a key role of PI4KA in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4, 5)P2-dependent calcium signaling.

Multi-image colocalization and its statistical significance.

Accurately localizing molecules within the cell is one of main tasks of modern biology, and colocalization analysis is one of its principal and most often used tools. Despite this popularity, interpretation is often uncertain because colocalization between two or more images is rarely analyzed to determine whether the observed values could have occurred by chance. To address this, we have developed a robust methodology, based on Monte Carlo randomization, to measure the statistical significance of a colocalization. The method works with voxel-based, intensity-based, object-based, and nearest-neighbor metrics. We extend all of these to measure colocalization in images with three colors. We also introduce three new metrics; blob colocalization, where the blob consists of a local maximum surrounded by a three-dimensional group of voxels; cluster diameter, to measure the clustering of fluorophores in three or more images; and the intercluster distance to measure the distance between these clusters. The robustness of these metrics was tested by varying the image thresholds over a broad range, which produced no change in the statistical significance of the colocalizations. A comparison of blob colocalization with voxel and Manders colocalization metrics shows that the different measures produce consistent results with similar values for significance and nonsignificance. Using our methodology, we are able to determine not only whether the labeled molecules colocalize with a probability greater than chance, but also whether they are sequestrated into different compartments. The program, written in C++, is freely available as source, as well as in a Linux version.

An integrated model of electrical spiking, bursting, and calcium oscillations in GnRH neurons.

The plasma membrane electrical activities of neurons that secrete gonadotropin-releasing hormone (GnRH) have been studied extensively. A couple of mathematical models have been developed previously to explain different aspects of these activities. The goal of this article is to develop a single model that accounts for the previously modeled experimental results and some more recent results that have not been accounted for. The latter includes two types of membrane potential bursting mechanisms and their associated cytosolic calcium oscillations. One bursting mechanism has not been reported in experiments and is thus regarded as a model prediction. Although the model is mainly based on data collected in immortalized GnRH cell lines, it is capable of explaining some properties of GnRH neurons observed in several other preparations including mature GnRH neurons in hypothalamic slices. We present a spatial model that incorporates a detailed description of calcium dynamics in a three-dimensional cell body with the ion channels evenly distributed on the cell surface. A phenomenological reduction of the spatial model into a simplified form is also presented. The simplified model will facilitate the study of the roles of plasma membrane electrical activities in the pulsatile release of GnRH.

Divergent expression patterns of pituitary gonadotropin subunit and GnRH receptor genes to continuous GnRH in vitro and in vivo.

Continuous, as opposed to pulsatile, delivery of hypothalamic gonadotropin-releasing hormone (GnRH) leads to a marked decrease in secretion of pituitary gonadotropins LH and FSH and impairment of reproductive function. Here we studied the expression profile of gonadotropin subunit and GnRH receptor genes in rat pituitary in vitro and in vivo to clarify their expression profiles in the absence and continuous presence of GnRH. Culturing of pituitary cells in GnRH-free conditions downregulated Fshb, Cga, and Gnrhr expression, whereas continuous treatment with GnRH agonists upregulated Cga expression progressively and Gnrhr and Fshb expression transiently, accompanied by a prolonged blockade of Fshb but not Gnrhr expression. In contrast, Lhb expression was relatively insensitive to loss of endogenous GnRH and continuous treatment with GnRH, probably reflecting the status of Egr1 and Nr5a1 expression. Similar patterns of responses were observed in vivo after administration of a GnRH agonist. However, continuous treatment with GnRH stimulated LH secretion in vitro and in vivo, leading to decrease in LH cell content despite high basal Lhb expression. These data suggest that blockade of Fshb expression and depletion of the LH secretory pool are two major factors accounting for weakening of the gonadotroph secretory function during continuous GnRH treatment.

Cell Type- and Sex-Dependent Transcriptome Profiles of Rat Anterior Pituitary Cells.

Understanding the physiology and pathology of an organ composed of a variety of cell populations depends critically on genome-wide information on each cell type. Here, we report single-cell transcriptome profiling of over 6,800 freshly dispersed anterior pituitary cells from postpubertal male and female rats. Six pituitary-specific cell types were identified based on known marker genes and characterized: folliculostellate cells and hormone-producing corticotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. Also identified were endothelial and blood cells from the pituitary capillary network. The expression of numerous developmental and neuroendocrine marker genes in both folliculostellate and hormone-producing cells supports that they have a common origin. For several genes, the validity of transcriptome analysis was confirmed by qRT-PCR and single cell immunocytochemistry. Folliculostellate cells exhibit impressive transcriptome diversity, indicating their major roles in production of endogenous ligands and detoxification enzymes, and organization of extracellular matrix. Transcriptome profiles of hormone-producing cells also indicate contributions toward those functions, while also clearly demonstrating their endocrine function. This survey highlights many novel genetic markers contributing to pituitary cell type identity, sexual dimorphism, and function, and points to relationships between hormone-producing and folliculostellate cells.

Common and diverse elements of ion channels and receptors underlying electrical activity in endocrine pituitary cells.

The pituitary gland contains six types of endocrine cells defined by hormones they secrete: corticotrophs, melanotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. All these cell types are electrically excitable, and voltage-gated calcium influx is the major trigger for their hormone secretion. Along with hormone intracellular content, G-protein-coupled receptor and ion channel expression can also be considered as defining cell type identity. While many aspects of the developmental and activity dependent regulation of hormone and G-protein-coupled receptor expression have been elucidated, much less is known about the regulation of the ion channels needed for excitation-secretion coupling in these cells. We compare the spontaneous and receptor-controlled patterns of electrical signaling among endocrine pituitary cell types, including insights gained from mathematical modeling. We argue that a common set of ionic currents unites these cells, while differential expression of another subset of ionic currents could underlie cell type-specific patterns. We demonstrate these ideas using a generic mathematical model, showing that it reproduces many observed features of pituitary electrical signaling. Mapping these observations to the developmental lineage suggests possible modes of regulation that may give rise to mature pituitary cell types.

Lamina propria and olfactory bulb ensheathing cells exhibit differential integration and migration and promote differential axon sprouting in the lesioned spinal cord.

Olfactory bulb-derived (central) ensheathing cell (OB OEC) transplants have shown significant promise in rat models of spinal cord injury, prompting the use of lamina propria-derived (peripheral) olfactory ensheathing cells (LP OECs) in both experimental and clinical trials. Although derived from a common embryonic precursor, both sources of OECs reside in different nervous system compartments postnatally, and their ability to promote regeneration and efficacy after transplantation may differ depending on both their source and mode of transplantation. Here, we have purified green fluorescent protein-expressing LP and OB OECs, assayed their biological differences in vitro, and transplanted them acutely either directly into or rostral and caudal to a dorsolateral funiculus crush. LP and OB OECs exhibit multiple morphological and antigenic similarities in vitro, and, after transplantation, they both attenuate lesion and cavity formation and promote angiogenesis, endogenous Schwann cell infiltration, and axonal sprouting. However, an increased mitotic rate and migratory ability of LP OECs in vitro was reflected in vivo by their superior ability to migrate within the spinal cord, reduce cavity formation and lesion size, and differentially stimulate outgrowth of axonal subpopulations compared with OB OECs. An undesired behavior (autotomy) was also significantly enhanced by LP OEC, over OB OEC, transplantation. These results suggest that LP and OB OECs exhibit intrinsic biological differences that, after transplantation into the lesioned CNS, result in differences in postlesion spinal cord neuropathology and anatomical and behavioral regeneration outcomes that also vary depending on direct versus rostrocaudal transplantation.

Direct stimulatory effects of oxytocin in female rat gonadotrophs and somatotrophs in vitro: comparison with lactotrophs.

The peptide oxytocin (OT) is secreted by hypothalamic neurons and exerts numerous actions related to reproduction. OT stimulation of prolactin secretion in female rats is important during the estrous cycle, pregnancy, and lactation. Here we report that OT also stimulates transients of intracellular Ca(2+) concentration in somatotrophs and gonadotrophs as well as the release of GH and LH in a dose-dependent manner with EC50 values that closely correspond to the ligand affinity of the OT receptor (OTR). Remarkably, the hormone-releasing effect of OT in these two cell types is 2 orders of magnitude more sensitive than that in lactotrophs. The specific OTR agonist [Thr(4),Gly(7)]-oxytocin acutely stimulated the release of LH, GH, and prolactin from female rat pituitary cells in primary culture and increased intracellular Ca(2+) concentration in gonadotrophs, somatotrophs, and lactotrophs. In these three cell types, the effects on hormone release and intracellular Ca(2+) of both OT and [Thr(4),Gly(7)]oxytocin were abolished by the specific OT receptor antagonist desGly-NH2-d(CH2)5[D-Tyr(2),Thr(4)]OVT but not by the highly selective vasopressin V1a receptor antagonist, d(CH2)5[Tyr(Me)(2),Dab(5)]AVP. Furthermore, 10 nM arginine vasopressin stimulated LH and GH release comparably with a dose of OT that was at least 10 times lower. Finally, the presence of the OTR-like immunoreactivity could be observed in all three cell types. Taken together, these results show that OT directly stimulates gonadotrophs, somatotrophs, and lactotrophs through OT receptors and suggest that OT signaling may serve to coordinate the release of different pituitary hormones during specific physiological conditions.

Interpreting frequency responses to dose-conserved pulsatile input signals in simple cell signaling motifs.

Many hormones are released in pulsatile patterns. This pattern can be modified, for instance by changing pulse frequency, to encode relevant physiological information. Often other properties of the pulse pattern will also change with frequency. How do signaling pathways of cells targeted by these hormones respond to different input patterns? In this study, we examine how a given dose of hormone can induce different outputs from the target system, depending on how this dose is distributed in time. We use simple mathematical models of feedforward signaling motifs to understand how the properties of the target system give rise to preferences in input pulse pattern. We frame these problems in terms of frequency responses to pulsatile inputs, where the amplitude or duration of the pulses is varied along with frequency to conserve input dose. We find that the form of the nonlinearity in the steady state input-output function of the system predicts the optimal input pattern. It does so by selecting an optimal input signal amplitude. Our results predict the behavior of common signaling motifs such as receptor binding with dimerization, and protein phosphorylation. The findings have implications for experiments aimed at studying the frequency response to pulsatile inputs, as well as for understanding how pulsatile patterns drive biological responses via feedforward signaling pathways.